Recombinant DNA constructs and methods for controlling gene expression

ABSTRACT

The present invention provides molecular constructs and methods for use thereof, including constructs including heterologous miRNA recognition sites, constructs for gene suppression including a gene suppression element embedded within an intron flanked on one or on both sides by non-protein-coding sequence, constructs containing engineered miRNA or miRNA precursors, and constructs for suppression of production of mature microRNA in a cell. Also provided are transgenic plant cells, plants, and seeds containing such constructs, and methods for their use. The invention further provides transgenic plant cells, plants, and seeds containing recombinant DNA for the ligand-controlled expression of a target sequence, which may be endogenous or exogenous. Also disclosed are novel miRNAs and miRNA precursors from crop plants including maize and soy.

PRIORITY CLAIMS AND INCORPORATION OF SEQUENCE LISTINGS

This is a divisional application of U.S. patent application Ser. No.11/303,745, filed 15 Dec. 2005, which claims the benefit of priority toU.S. Provisional Patent Applications 60/638,256, which was filed on 21Dec. 2004, 60/639,094, which was filed on 24 Dec. 2004, 60/701,124,which was filed on 19 Jul. 2005, 60/711,834, which was filed on 26 Aug.2005, 60/720,005, which was filed on 24 Sep. 2005, 60/726,106, which wasfiled on 13 Oct. 2005, and 60/736,525, which was filed on 14 Nov. 2005,incorporated by reference in their entirety herein. The sequence listingthat is contained in the file named “38-15(53429)100.rpt” which is 97kilobytes (measured in operating system MS-Windows), created on 29 Sep.2006, and located in computer readable form on a compact disk (CD-R), isfiled herewith and incorporated herein by reference. The sequencelisting contained in the file named “38-15(53429)C.rpt”, which is 97kilobytes (measured in MS-Windows), located in computer readable form ona compact disk created on 28 Sep. 2006, and filed on 29 Sep. 2006 as areplacement sequence listing for U.S. patent application Ser. No.11/303,745, which was filed on 15 Dec. 2005, is incorporated byreference in its entirety herein. The sequence listings contained in thefiles “53429A.ST25.txt” (file size of 15 kilobytes, recorded on 21 Dec.2004, and filed with U.S. Provisional Application 60/638,256 on 21 Dec.2004), “38-21(53709)B.ST25.txt” (file size of 4 kilobytes, recorded on23 Dec. 2004, and filed with U.S. Provisional Application 60/639,094 on24 Dec. 2004), “38-15(53429)B.rpt” (file size of 7 kilobytes, recordedon 19 Jul. 2005, filed with U.S. Provisional Application 60/701,124 on19 Jul. 2005), “38-15(54068)A.rpt” (file size of 6 kilobytes, recordedon 26 Aug. 2005, filed with U.S. Provisional Application 60/711,834 on26 Aug. 2005), “38-21(54176)A.rpt” (file size of 29 kilobytes, recordedon 23 Sep. 2005, and filed with U.S. Provisional Application 60/720,005on 24 Sep. 2005), and “38-21(54232)A.rpt” (file size of 61 kilobytes,recorded on 12 Oct. 2005, and filed with U.S. Provisional Application60/726,106 on 13 Oct. 2005) are incorporated by reference in theirentirety herein.

FIELD OF THE INVENTION

The present invention discloses molecular constructs and methods for thecontrol of gene expression, for example, gene suppression in plants orin plant pests or pathogens or suppressing expression of a target RNA ina specific cell. Also disclosed are transgenic eukaryotes, includingtransgenic plant cells, plants, and seeds, whose genome includesmolecular constructs for controlling expression of an endogenous or anexogenous gene.

BACKGROUND OF THE INVENTION

Nucleic acid aptamers include DNA or RNA sequences that can recognizeand specifically bind, often with high affinity, a particular moleculeor ligand. See, for example, reports describing in vitro aptamerselection by Tuerk and Gold (1990) Science, 249:505-510, Ellington andSzostak (1990) Nature, 346:818-822, and Ellington and Szostak (1992)Nature, 355:850-852, as well as Jenison et al. (1994) Science,263:1425-1429, which demonstrated the ability of an RNA aptamer todistinguish between theophylline and caffeine (which differ by a singlemethyl group) by four orders of magnitude. Similar to antibodies thatbind specific antigens or receptors that bind specific molecules,aptamers are useful alone, to bind to a specific ligand (see, forexample, Shi et al. (1999) Proc. Natl. Acad. Sci. USA, 96:10033-10038,which describes a multivalent RNA aptamer effective as a proteinantagonist), and in combination, e. g., as a molecular “escort” fordelivery of an agent to a specific location, cell, or tissue (see, forexample, Hicke and Stephens (2000) J. Clin. Investigation, 106:923-928)or as part of a riboswitch. Riboswitches are complex folded RNAsequences including an aptamer domain for a specific ligand. Naturallyoccurring riboswitches have been found mainly in bacteria, and morerecently in fungi (Kubodera et al. (2003) FEBS Lett., 555:516-520) andplants (Sudarsan et al. (2003) RNA, 9:644-647, which is incorporated byreference). Many riboswitches contain conserved domains within species(Barrick et al, (2004) Proc. Natl. Acad. Sci. USA, 101:6421-6426, whichis incorporated by reference). Riboswitches that act in a “cis” fashion(i. e., that control expression of an operably linked sequence) areknown to occur in the non-coding regions of mRNAs in prokaryotes, wherethey control gene expression by harnessing allosteric structural changescaused by ligand binding. For a review of “cis” riboswitches, see Mandaland Breaker (2004a) Nature Rev. Mol. Cell Biol., 5:451-463, which isincorporated by reference. Riboswitches that act in a “trans” fashion(i. e., that control expression of a sequence not operably linked to theriboswitch) have also been designed, see, for example, Bayer and Smolke(2005) Nature Biotechnol., 23:337-343, which is incorporated byreference.

Most known naturally occurring riboswitches are “off” switches, whereinthe default state is “on” (i. e., the gene under the riboswitch'scontrol is expressed), and ligand binding turns the gene “off”. Inprokaryotes, these riboswitches have been found mainly in the 5′untranslated region (5′ UTR) of mRNAs encoding biosynthesis genes; ineukaryotes, riboswitches have been found in the 3′ untranslated region(3′ UTR) or within introns (Sudarsan et al. (2003) RNA, 9:644-647;Templeton and Moorhead (2004) Plant Cell, 16:2252-2257). When anincreased concentration of a particular metabolite or ligand is “sensed”by the riboswitch (bound by the aptamer domain), the riboswitch“switches off” gene expression through transcription termination and/ortranslation attenuation; see, for example, FIG. 2 in Mandal and Breaker(2004a) Nature Rev. Mol. Cell Biol., 5:451-463 and FIG. 4 in Sudarsan etal. (2003) RNA, 9:644-647.

At least two types of “on” riboswitches have been reported, wherein thedefault state is “off” and ligand binding turns the gene “on”.Expression of ydhL, encoding a purine exporter, is turned on by adeninebinding to the ydhL aptamer; see Mandal and Breaker (2004b) NatureStruct. Mol. Biol., 11:29-35). Similarly lysine “on” riboswitches havebeen proposed to activate the expression of lysine exporter ordegradation genes; see Rodionov et al. (2003) Nucleic Acids Res.,31:6748-6757. There are also lysine “off” riboswitches that control theexpression of lysine biosynthesis genes; see Sudarsan et al. (2003)Genes Dev., 17:2688-2697.

A typical riboswitch is composed of an aptamer domain that remainslargely conserved, and a regulatory domain that can vary more widelyduring evolution. In a non-limiting example, the coenzyme-B₁₂ riboswitchcontrols gene expression by two main mechanisms, as dictated by thearchitecture of the regulatory domain (see FIG. 2 in Mandal and Breaker(2004a) Nature Rev. Mol. Cell Biol., 5:451-463). If the regulatorydomain contains a “terminator stem”, the binding of coenzyme-B₁₂ to itsaptamer triggers transcriptional termination. If the expression platformcontains an “anti-ribosome binding site stem”, the binding ofcoenzyme-B₁₂ to its aptamer triggers translational attenuation. In someinstances, it is believed that transcription and translation can becontrolled simultaneously.

The present invention provides a novel transgenic plant having in itsgenome recombinant DNA that transcribes to at least one RNA aptamer towhich a ligand binds, and can further include at least one regulatoryRNA domain capable of regulating the target sequence. Depending on thedesign of the recombinant DNA, the regulatory RNA can act “in trans” or“in cis” in the transgenic plants to control expression of an endogenousor of an exogenous target sequence, and the ligand can be exogenous orendogenous. Transgenic plants of the invention are preferably stablytransgenic plants in which a desired trait, or an altered trait, isachieved in the transgenic plant (or in a seed or progeny plant of thetransgenic plant) according to whether or not the ligand is bound to theaptamer and the resulting expression (or suppression) of the targetsequence.

Current methods to suppress a gene include, for example, the use ofantisense, co-suppression, and RNA interference. Anti-sense genesuppression in plants is described by Shewmaker et al. in U.S. Pat. Nos.5,107,065, 5453,566, and 5,759,829. Gene suppression in bacteria usingDNA which is complementary to mRNA encoding the gene to be suppressed isdisclosed by Inouye et al. in U.S. Pat. Nos. 5,190,931, 5,208,149, and5,272,065. RNA interference or double-stranded RNA-mediated genesuppression has been described by, e. g., Redenbaugh et al. in “SafetyAssessment of Genetically Engineered Fruits and Vegetables”, CRC Press,1992; Chuang et al. (2000) PNAS, 97:4985-4990; Wesley et al. (2001)Plant J., 27:581-590.

The efficiency of anti-sense gene suppression is typically low.Redenbaugh et al. in “Safety Assessment of Genetically Engineered Fruitsand Vegetables”, CRC Press, 1992, report a transformation efficiencyranging from 1% to 20% (page 113) for tomato transformed with aconstruct designed for anti-sense suppression of the polygalacturonasegene. Chuang et al. reported in PNAS, (2000) 97:4985-4990 thatanti-sense constructs, sense constructs, and constructs where anti-senseand sense DNA are driven by separate promoters had either no, or weak,genetic interference effects as compared to potent and specific geneticinterference effects from dsRNA constructs (see FIG. 1 and Table 1,PNAS, (2000) 97:4985-4990). See also Wesley et al. who report in ThePlant Journal, (2001) 27:581-590, e. g., at Table 1, the comparativeefficiency of hairpin RNA, sense constructs, and anti-sense constructsat silencing a range of genes in a range of plant species with a clearindication that the efficiency for anti-sense constructs is typicallyabout an order of magnitude lower than the efficiency for hairpin RNA.

Matzke et al. in Chapter 3 (“Regulation of the Genome by double-strandedRNA”) of “RNAi—A Guide to Gene Silencing”, edited by Hannon, Cold SpringHarbor Laboratory Press, 2003, discuss the use of polyadenylationsignals in promoter inverted repeat constructs. At page 58, they statethat “the issue of whether to put polyadenylation signals in promoterinverted repeat constructs is unsettled because the nature of the RNAtriggering RdDM [RNA-directed DNA methylation] is unresolved. Dependingon whether short RNA or dsRNA is involved in RdDM, the decision toinclude a polyadenylation site might differ depending on theexperimental system used. If dsRNA is involved in RdDM, then apolyadenylation signal is not required because dsRNA forms rapidly byintramolecular folding when the entire inverted repeat is transcribed.Indeed, nonpolyadenylated dsRNAs might be retained in the nucleus andinduce RdDM more efficiently than polyadenylated dsRNAs. Matzke et al.continue: “If short RNAs guide homologous DNA methylation, then thesituation in plants and mammals differ. In plants, which probablypossess a nuclear form of Dicer, non-polyadenylated dsRNAs would stillbe optimal because they should feed preferentially into a nuclearpathway for dsRNA processing.”

Carmichael et al. in U.S. Pat. Nos. 5,908,779 and 6,265,167 disclosemethods and constructs for expressing and accumulating anti-sense RNA inthe nucleus using a construct that comprises a promoter, anti-sensesequences, and sequences encoding a cis-or trans-ribozyme. Thecis-ribozyme is incorporated into the anti-sense construct in order togenerate 3′ ends independently of the polyadenylation machinery andthereby inhibit transport of the RNA molecule to the cytoplasm.Carmichael demonstrated the use of the construct in mouse NIH 3T3 cells.

Various other nucleic acid constructs and methods for gene suppressionhave been described in recent publications. Shewmaker et al. (U.S. Pat.No. 5,107,565) disclose constructs for gene silencing that can containtwo or more repetitive anti-sense sequence in tandem for modulating oneor more genes. Resistance to a virus was achieved in a transgenic plantby use of a transgene containing a direct repeat of the virus's movementprotein (Sijen et al. (1996) Plant Cell, 8:2277-2294). Another reportdemonstrated that nucleic acid constructs containing a promoter, aterminator, and direct or interrupted tandem repeats of either sense oranti-sense sequences, could induce gene silencing in plants (Ma andMitra (2002) Plant J., 31:37-49. The expression of1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was downregulated intransgenic tomatoes containing a nucleic acid construct including adirect repeat of the ACC oxidase 5′ untranslated region sequence in theanti-sense orientation (Hamilton et al. (1998) Plant J., 15:737-346).Waterhouse and Wang (U.S. Patent Application Publication 2003/0165894)disclose a method for reducing phenotypic expression using nucleic acidconstructs that transcribe to aberrant RNAs including unpolyadenylatedRNAs. Clemente et al. disclose nucleic acid constructs including senseor anti-sense sequences lacking a normal 3′ untranslated region andoptionally including a ribozyme, that transcribe to unpolyadenylatedRNA. All of the patents cited in this paragraph are incorporated byreference in their entirety herein.

DNA is either coding (protein-coding) DNA or non-coding DNA. Non-codingDNA includes many kinds of non-translatable (non-protein-coding)sequence, including 5′ untranslated regions, promoters, enhancers, orother non-coding transcriptional regions, 3′ untranslated regions,terminators, and introns. The term “intron” is generally applied tosegments of DNA (or the RNA transcribed from such segments) that arelocated between exons (protein-encoding segments of the DNA), wherein,during maturation of the messenger RNA, the introns present areenzymatically “spliced out” or removed from the RNA strand by acleavage/ligation process that occurs in the nucleus in eukaryotes. Linet al. (2003) Biochem. Biophys. Res. Comm., 310:754-760, and Lin et al.U.S. Patent Application Publications 2004/0106566 and 2004/0253604,which are incorporated by reference in their entirety herein, disclosemethods for inducing gene silencing using nucleic acid constructscontaining a gene silencing molecule (sense or anti-sense or both)within an intron flanked by multiple protein-coding exons, wherein, uponsplicing and removal of the intron, the protein-coding exons are linkedto form a mature mRNA encoding a protein with desired function and thegene silencing molecule is released.

However, apart from introns found between protein-encoding exons, thereare other non-coding DNA sequences that can be spliced out of a maturingmessenger RNA. One example of these are spliceable sequences that thathave the ability to enhance expression in plants (in some cases,especially in monocots) of the downstream coding sequence; thesespliceable sequences are naturally located in the 5′ untranslated regionof some plant genes, as well as in some viral genes (e. g., the tobaccomosaic virus 5′ leader sequence or “omega” leader described as enhancingexpression in plant genes by Gallie and Walbot (1992) Nucleic AcidsRes., 20:4631-4638). These spliceable sequences or “expression-enhancingintrons” can be artificially inserted in the 5′ untranslated region of aplant gene between the promoter but before any protein-coding exons. Forexample, it was reported that inserting a maize alcohol dehydrogenase(Zm-Adh1) or Bronze-1 expression-enhancing intron 3′ to a promoter (e.g., Adh1, cauliflower mosaic virus 35S, or nopaline synthase promoters)but 5′ to a protein-coding sequence (e. g., chloramphenicolacetyltransferase, luciferase, or neomycin phosphotransferase II)greatly stimulated expression of the protein (Callis et al. (1987) GenesDev., 1:1183-1200). The Adh1 intron greatly stimulated expression of areporter gene (Mascarenkas et al. (1990) Plant Mol. Biol., 15:913-920).Cis-acting elements that increase transcription of a downstream codingsequence in transformed plant cells were reported to occur in the 5′untranslated region of the rice actin 1 (Os-Act1) gene (Wang et al.(1992) Mol. Cell Biol., 12:3399-3406). The rice Act1gene was furthercharacterized to contain a 5′ expression-enhancing intron that islocated upstream of the first protein-coding exon and that is essentialfor efficient expression of coding sequence under the control of theAct1 promoter (McElroy et al. (1990) Plant Cell, 2:163-171). TheShrunken-1 (Sh-1) intron was reported to give about 10 times higherexpression than constructs containing the Adh-1 intron (Vasil et al.(1989) Plant Physiol., 91:1575-1579). The maize sucrose synthase intron,when placed between a promoter and the firs protein-coding exon, alsoincreases expression of the encoded protein, and splicing of the intronis required for this enhanced expression to occur (Clancy and Hannah(2002) Plant Physiol., 130:918-929). Expression-enhancing introns havealso been characterized for heat shock protein 18 (hsp18) (Silva et al.(1987) J. Cell Biol., 105:245) and the 82 kilodalton heat shock protein(hsp82) (Semrau et al. (1989) J. Cell Biol., 109, p. 39A, and Mettler etal. (May 1990) N.A.T.O. Advanced Studies Institute on Molecular Biology,Elmer, Bavaria). U.S. Pat. Nos. 5,593,874 and 5,859,347 describeimproved recombinant plant genes including a chimeric plant gene with anexpression-enhancing intron derived from the 70 kilodalton maize heatshock protein (hsp70) in the non-translated leader positioned 3′ fromthe gene promoter and 5′ from the first protein-coding exon. All of thepatents and publications cited in this paragraph are incorporated byreference herein.

The present inventors have found that, unexpectedly, introns can beutilized to deliver a gene suppression element in the absence of anyprotein-coding exons (coding sequence). In the present invention, anintron, such as an expression-enhancing intron (preferred in certainembodiments), is interrupted by embedding within the intron a genesuppression element, wherein, upon transcription, the gene suppressionelement is excised from the intron to function in suppressing a targetgene. Thus, no protein-coding exons are required to provide the genesuppressing function of the recombinant DNA constructs disclosed herein.

MicroRNAs (miRNAs) are non-protein coding RNAs, generally of betweenabout 19 to about 25 nucleotides (commonly about 20-24 nucleotides inplants), that guide cleavage in trans of target transcripts, negativelyregulating the expression of genes involved in various regulation anddevelopment pathways (Bartel (2004) Cell, 116:281-297). In some cases,miRNAs serve to guide in-phase processing of siRNA primary transcripts(see Allen et al. (2005) Cell, 121:207-221, which is incorporated hereinby reference).

Some microRNA genes (MIR genes) have been identified and made publiclyavailable in a database ('miRBase”, available on line atmicrorna.sanger.ac.uk/sequences). Additional MIR genes and mature miRNAsare also described in U.S. Patent Application Publications 2005/0120415and 2005/144669A1, which is incorporated by reference herein. MIR geneshave been reported to occur in inter-genic regions, both isolated and inclusters in the genome, but can also be located entirely or partiallywithin introns of other genes (both protein-coding andnon-protein-coding). For a recent review of miRNA biogenesis, see Kim(2005) Nature Rev. Mol. Cell Biol., 6:376-385. Transcription of MIRgenes can be, at least in some cases, under promotional control of a MIRgene's own promoter. MIR gene transcription is probably generallymediated by RNA polymerase II (see, e. g., Aukerman. and Sakai (2003)Plant Cell, 15:2730-2741; Parizotto et al. (2004) Genes Dev.,18:2237-2242), and therefore could be amenable to gene silencingapproaches that have been used in other polymerase II-transcribed genes.The primary transcript (which can be polycistronic) termed a“pri-miRNA”, a miRNA precursor molecule that can be quite large (severalkilobases) and contains one or more local double-stranded or “hairpin”regions as well as the usual 5′ “cap” and polyadenylated tail of anmRNA. See, for example, FIG. 1 in Kim (2005) Nature Rev. Mol. CellBiol., 6:376-385.

In animal cells, this pri-miRNA is believed to be “cropped” by thenuclear RNase III Drosha to produce a shorter miRNA precursor moleculeknown as a “pre-miRNA”. Following nuclear processing by Drosha,pre-miRNAs are exported to the nucleus where the enzyme Dicer generatesthe short, mature miRNAs. See, for example, Lee et al. (2002) EMBOJournal, 21:4663-4670; Reinhart et al. (2002) Genes & Dev.,16:161611626; Lund et al. (2004) Science, 303:95-98; and Millar andWaterhouse (2005) Funct. Integr Genomics, 5:129-135, which areincorporated by reference herein. In contrast, in plant cells, microRNAprecursor molecules are believed to be largely processed in the nucleus.Whereas in animals both miRNAs and siRNAs are believed to result fromactivity of the same DICER enzyme, in plants miRNAs and siRNAs areformed by distinct DICER-like (DCL) enzymes, and in Arabidopsis anuclear DCL enzyme is believed to be required for mature miRNA formation(Xie et al. (2004) PLoS Biol., 2:642-652, which is incorporated byreference herein). Additional reviews on microRNA biogenesis andfunction are found, for example, in Bartel (2004) Cell, 116:281-297;Murchison and Hannon (2004) Curr. Opin. Cell Biol., 16:223-229; andDugas and Bartel (2004) Curr. Opin. Plant Biol., 7:512-520. MicroRNAscan thus be described in terms of RNA (e. g., RNA sequence of a maturemiRNA or a miRNA precursor RNA molecule), or in terms of DNA (e. g., DNAsequence corresponding to a mature miRNA RNA sequence or DNA sequenceencoding a MIR gene or fragment of a MIR gene or a miRNA precursor).

MIR gene families appear to be substantial, estimated to account for 1%of at least some genomes and capable of influencing or regulatingexpression of about a third of all genes (see, for example, Tomari etal. (2005) Curr. Biol., 15:R61-64; G. Tang (2005) Trends Biochem. Sci.,30:106-14; Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385). BecausemiRNAs are important regulatory elements in eukaryotes, includinganimals and plants, transgenic suppression of miRNAs could, for example,lead to the understanding of important biological processes or allow themanipulation of certain pathways useful, for example, inbiotechnological applications. For example, miRNAs are involved inregulation of cellular differentiation, proliferation and apoptosis, andare probably involved in the pathology of at least some diseases,including cancer, where miRNAs may function variously as oncogenes or astumor suppressors. See, for example, O'Donnell et al. (2005) Nature,435:839-843; Cai et al. (2005) Proc. Natl. Acad. Sci. USA,102:5570-5575; Morris and McManus (2005) Sci. STKE, pe41 (availableonline at stke.sciencemag.org/cgi/reprint/sigtrans;2005/297/pe41.pdf).MicroRNA (MIR) genes have identifying characteristics, includingconservation among plant species, a stable foldback structure, andprocessing of a specific miRNA/miRNA* duplex by Dicer-like enzymes(Ambros et al. (2003) RNA, 9:277-279). These characteristics have beenused to identify miRNAs and their corresponding genes in plants (Xie etal. (2005) Plant Physiol., 138:2145-2154; Jones-Rhoades and Bartel(2004) Mol. Cell, 14:787-799; Reinhart et al. (2002) Genes Dev.,16:1616-1626; Sunkar and Zhu (2004) Plant Cell, 16:2001-2019). Publiclyavailable microRNA genes are catalogued at miRBase (Griffiths-Jones etal. (2003) Nucleic Acids Res., 31:439-441).

MiRNAs have been found to be expressed in very specific cell types inArabidopsis (see, for example, Kidner and Martienssen (2004) Nature,428:81-84, Millar and Gubler (2005) Plant Cell, 17:705-721). Suppressioncan be limited to a side, edge, or other division between cell types,and is believed to be required for proper cell type patterning andspecification (see, for example, Palatnik et al. (2003) Nature,425:257-263). Suppression of a GFP reporter gene containing anendogenous miR171 recognition site was found to limit expression tospecific cells in transgenic Arabidopsis (Parizotto et al. (2004) GenesDev., 18:2237-2242). Recognition sites of miRNAs have been validated inall regions of an mRNA, including the 5′ untranslated region, codingregion, and 3′ untranslated region, indicating that the position of themiRNA target site relative to the coding sequence may not necessarilyaffect suppression (see, for example, Jones-Rhoades and Bartel (2004).Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell, 110:513-520, Allen etal. (2004) Nat. Genet., 36:1282-1290, Sunkar and Zhu (2004) Plant Cell,16:2001-2019).

The invention provides novel recombinant DNA constructs and methods foruse thereof for suppression of production of mature miRNA in a cell,where the constructs are designed to target at least one miRNA precursoror at least one promoter of a miRNA precursor. Using constructs of theinvention, suppression of production of mature miRNA can occur in thenucleus or in the cytoplasm or in both. In plants, microRNA precursormolecules are believed to be largely processed in the nucleus. Thus, inmany preferred embodiments of the recombinant DNA construct of theinvention, particularly (but not limited to) embodiments where thesuppression occurs in a plant cell, suppression preferably occurs whollyor substantially in the nucleus. Another potential advantage of theinvention is that miRNA precursors (especially pri-miRNAs, and to alesser extent pre-miRNAs) offer substantially larger target sequencesthan does a mature miRNA.

In a preferred embodiment, the constructs and methods of the inventionare designed to target nuclear-localized miRNA precursors (such aspri-miRNAs and pre-miRNA) prior to their export from the nucleus; suchembodiments provide an advantage over conventional gene suppressionconstructs (e. g., containing inverted repeats) that typically result inaccumulation of dsRNA in the cytoplasm. In such embodiments, recombinantDNA constructs of the invention include a gene suppression elementdesigned to remain in the nucleus after transcription, for example, agene suppression element that is transcribed to RNA lacking functionalnuclear export signals. Such embodiments are particularly preferred foruse, e. g., in plants, where processing of miRNA is believed to occurlargely in the nucleus. In one preferred embodiment of the invention,the recombinant DNA construct includes a suppression element (e. g., oneor more inverted repeats, anti-sense sequence, tandem repeats, or othersuppression elements) embedded within a spliceable intron. The resultingsuppression transcript remains in the nucleus, preferably resulting inthe nuclear degradation of the target pri-miRNA or pre-miRNA, oralternatively, resulting in transcriptional silencing of a target MIRgene promoter, which, in turn, reduces the accumulation of the maturemiRNA.

In other embodiments, recombinant DNA constructs of the inventioninclude a suppression element transcribable to RNA that is exported fromthe nucleus to the cytoplasm, where, for example, the transcribed andexported RNA targets a cytoplasmic pre-miRNA. Such embodiments areparticularly useful where miRNA processing at least partly occurs in thecytoplasm, e. g., in animal cells. In such embodiments, the suppressionelement is preferably transcribed to RNA including functional nuclearexport signals.

In multicellular eukaryotes, including plants, microRNAs (miRNAs)regulate endogenous genes by a post-transcriptional cleavage mechanismin a cell-type specific manner. The invention further provides arecombinant DNA construct, and methods for the use thereof, wherein theconstruct includes transcribable DNA that transcribes to RNA including(a) at least one exogenous miRNA recognition site recognizable by amature miRNA expressed in a specific cell, and (b) target RNA to besuppressed in the specific cell, whereby said target RNA is expressed incells other than said specific cell. These constructs are useful forsuppressing expression of a target RNA in a specific cell of amulticellular eukaryote (but allowing expression in other cells),including transcribing in the multicellular eukaryote a recombinant DNAconstruct including a promoter operably linked to DNA that transcribesto RNA including: (a) at least one exogenous miRNA recognition siterecognizable by a mature miRNA expressed in a specific cell, and (b)target RNA to be suppressed in the specific cell, wherein the maturemiRNA guides cleavage of target RNA in the specific cell, wherebyexpression of the target RNA is suppressed in the specific cell relativeto its expression in cells lacking expression of the mature miRNA.

The present invention further provides novel mature miRNA sequences andMIR gene sequences from crop plants, including maize and soybean. Themature miRNAs processed from these genes belong to canonical familiesconserved across distantly related plant species. These MIR genes andtheir encoded mature miRNAs are useful, e. g., for modifyingdevelopmental pathways, e. g., by affecting cell differentiation ormorphogenesis (see, for example, Palatnik et al. (2003) Nature,425:257-263; Mallory et al. (2004) Curr. Biol., 14:1035-1046), to serveas sequence sources for engineered (non-naturally occurring) miRNAs thatare designed to target sequences other than the transcripts targetted bythe naturally occurring miRNA sequence (see, for example, Parizotto etal. (2004) Genes Dev., 18:2237-2242, and U.S. Patent ApplicationPublications 2004/3411A1, 2005/0120415, which are incorporated byreference herein), and to stabilize dsRNA. A MIR gene itself (or itsnative 5′ or 3′ untranslated regions, or its native promoter or otherelements involved in its transcription) is useful as a target sequencefor gene suppression (e. g., by methods of the present invention), wheresuppression of the miRNA encoded by the MIR gene is desired. Promotersof MIR genes can have very specific expression patterns (e. g.,cell-specific, tissue-specific, or temporally specific), and thus areuseful in recombinant constructs to induce such specific transcriptionof a DNA sequence to which they are operably linked.

SUMMARY OF THE INVENTION

The present invention discloses a transgenic plant cell, as well astransgenic plants and transgenic seed of such plants, having in itsgenome recombinant DNA for the ligand-controlled expression of a targetsequence. One aspect of this invention provides a transgenic plant cellhaving in its genome recombinant DNA including transcribable DNAincluding DNA that transcribes to an RNA aptamer capable of binding to aligand. In some embodiments of the invention, the recombinant DNAfurther includes at least one T-DNA border. In many embodiments, thetranscribable DNA further includes DNA that transcribes to regulatoryRNA capable of regulating expression of a target sequence, wherein theregulation of the target sequence is dependent on the conformation ofthe regulatory RNA, and the conformation of the regulatory RNA isallosterically affected by the binding state of the RNA aptamer.

Another aspect of the invention provides a method of reducing damage toa plant by an invertebrate pest or by a bacterial, fungal, or viralpathogen of said plant, including transcribing in the plant arecombinant DNA construct including transcribable DNA including DNA thattranscribes to an RNA aptamer capable of binding to a ligand, whereinthe ligand comprises at least part of a molecule endogenous to the pestor pathogen, and whereby binding of the RNA aptamer to the ligandreduces damage to the plant by the pest or pathogen, relative to damagein the absence of transcription of the recombinant DNA construct. Inparticularly preferred embodiments, the pest or pathogen is aninvertebrate pest of the plant, and the ligand includes at least part ofa molecule of the digestive tract lining of the invertebrate pest.

Another aspect of the invention provides a recombinant DNA constructincluding: (a) transcribable DNA including DNA that transcribes to anRNA aptamer capable of binding to a ligand; and (b) DNA sequence thattranscribes to double-stranded RNA flanking said transcribable DNA. Insome embodiments, the recombinant DNA construct further includes DNAthat transcribes to regulatory RNA capable of regulating expression of atarget sequence, wherein the regulation is dependent on the conformationof the regulatory RNA, and the conformation of the regulatory RNA isallosterically affected by the binding state of the RNA aptamer.

The present invention discloses recombinant DNA constructs forsuppression of at least one target gene, as well as methods for theiruse. In one aspect, the present invention provides a recombinant DNAconstruct for plant transformation including a first gene suppressionelement for suppressing at least one first target gene, wherein the genesuppression element is embedded in an intron, and wherein the intron islocated adjacent to at least one element selected from the groupconsisting of a promoter element and a terminator element. The constructcan optionally include at least one T-DNA border, a second genesuppression element, a gene expression element, or both. The inventionfurther provides transgenic plant cells and transgenic plants and seedsderived therefrom, containing such a recombinant DNA construct, and amethod for effecting gene suppression by expressing such a recombinantDNA construct in a transgenic plant.

In another aspect, the present invention provides a transgenic seedhaving in its genome a recombinant DNA construct for suppressing atleast one first target gene, including DNA capable of initiatingtranscription in a plant and operably linked to a first transcribableheterologous DNA, wherein said first transcribable heterologous DNA isembedded in an intron. The invention further provides a transgenic plantgrown from the transgenic seed, and methods for gene suppression or forconcurrent gene suppression and gene expression, that include growingsuch transgenic plants. A potential advantage of the use of constructsof this invention is avoidance of unintentional systemic spreading ofgene suppression.

Another aspect of the invention discloses recombinant DNA constructs andmethods for suppression of production of mature microRNA in a cell, forexample, by targetting for suppression a miRNA precursor or a promoterof a miRNA gene

In one aspect, the present invention provides a recombinant DNAconstruct for suppressing production of mature microRNA (miRNA) in acell, including a promoter element operably linked to a suppressionelement for suppression of at least one target microRNA precursor. Therecombinant DNA constructs include at least one suppression element forsuppression of at least one target microRNA precursor. The suppressionelement suppresses at least one target sequence selected from a targetsequence of the at least one target microRNA precursor, or a targetsequence of a promoter of the at least one target microRNA precursor, orboth.

In another aspect, the present invention provides a transgenic planthaving in its genome the recombinant DNA construct of the invention (i.e., a recombinant DNA construct for suppressing production of maturemicroRNA (miRNA) in a cell, including a promoter element operably linkedto a suppression element for suppression of at least one target microRNAprecursor), as well as seed and progeny of such transgenic plants.

In still another aspect, the present invention provides a method tosuppress expression of a target sequence in a plant cell, includingtranscribing in a plant cell a recombinant DNA construct including atranscribable engineered miRNA precursor, derived from the fold-backstructure of a maize or soybean MIR sequence or their complements,designed to suppress a target sequence, whereby expression of the targetsequence is suppressed relative to its expression in the absence oftranscription of the recombinant DNA construct.

In a further aspect, the present invention provides a recombinant DNAconstruct including a promoter operably linked to DNA that transcribesto RNA including (a) at least one exogenous miRNA recognition siterecognizable by a mature miRNA expressed in a specific cell, and (b)target RNA to be suppressed in the specific cell, whereby said targetRNA is expressed in cells other than said specific cell.

In yet another aspect, the present invention provides methods forsuppressing expression of a target RNA in a specific cell of amulticellular eukaryote, including transcribing in the multicellulareukaryote a recombinant DNA construct including a promoter operablylinked to DNA that transcribes to RNA including: (a) at least oneexogenous miRNA recognition site recognizable by a mature miRNAexpressed in a specific cell, and (b) target RNA to be suppressed in thespecific cell, wherein the mature miRNA guides cleavage of target RNA inthe specific cell, whereby expression of the target RNA is suppressed inthe specific cell relative to its expression in cells lacking expressionof the mature miRNA.

Other specific embodiments of the invention are disclosed in thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates DNA vectors as described in Example 1.Legend: pale grey regions labelled “e35X-Hsp70”: a chimeric promoterelement including an enhanced CaMV35S promoter linked to an enhancerelement (an intron from heat shock protein 70 of Zea mays, Pe35S-Hsp70intron); medium grey regions labeled “LUC”: DNA coding for fireflyluciferase; dark grey regions labeled “3′ nos”: a 3′UTR DNA fromAgrobacterium tumefaciens nopaline synthase gene. Vectors areconventionally depicted as transcribing from left (5′) to right (3′).Arrows indicate orientation of the luciferase segments as sense(arrowhead to right) or anti-sense (arrowhead to left).

FIG. 2 schematically illustrates DNA vectors as described in Example 2.Legend: pale grey regions labelled “e35s”: a chimeric promoter includingan enhanced CaMV35S promoter linked to an enhancer element (an intronfrom heat shock protein 70 of Zea mays, Pe35S-Hsp70 intron); medium greyregions labeled “GUS”: DNA coding for beta-glucuronidase; medium greyregions labeled “LUC”: DNA coding for firefly luciferase; dark greyregions labeled “3′ nos”: a 3′UTR DNA from Agrobacterium tumefaciensnopaline synthase gene. Vectors are conventionally depicted astranscribing from left (5′) to right (3′). Arrows indicate orientationof the luciferase segments as sense (arrowhead to right) or anti-sense(arrowhead to left).

FIG. 3 depicts results of the experiments described in Example 2. X-axisindicates the vectors (see FIG. 2) used. Y-axis values are given as thelogarithm of the ratio of logarithm of the ratio of firefly luciferaseto Renilla luciferase, “log(Fluc/Rluc)”; error bars are 95% confidenceintervals.

FIG. 4 is a schematic map of a plasmid including an enhanced anti-senseconstruct as described in Example 3.

FIG. 5A is a schematic map of a vector including an enhanced anti-senseconstruct and described in Example 4. The plasmid includes an aroA geneas an herbicidal selectable marker, and a recombinant DNA construct forenhanced anti-sense gene suppression, consisting of a seed-specificmaize L3 oleosin promoter operably linked to transcribable DNAconsisting of about 300 base pairs of a maize lysine ketoglutaratereductase (LKR) gene (LKR region of the lysine ketoglutaratereductase//saccharopine dehydrogenase gene, LKR/SDH) in an anti-senseorientation, wherein a functional polyadenylation site is absent in thistranscribable DNA, and left T-DNA border (LB) and right T-DNA border(RB) elements. FIG. 5B depicts a recombinant DNA construct of thepresent invention for gene suppression and described in Example 4,including left T-DNA border (LB) and right T-DNA border (RB) elements,and a promoter element operably linked to an intron (maize heat shockprotein 70 intron, I-Zm-hsp70) within which is embedded a first genesuppression element for suppressing at least one first target gene (inthis example, maize lysine ketoglutarate reductase/saccharopinedehydrogenase gene (LKR/SDH)). The first gene suppression element caninclude any gene suppression element as described above under theheading “Gene Suppression Elements” wherein the intron is locatedadjacent to the promoter element. In the specific, non-limitingembodiment depicted in FIG. 5B, the promoter element is anendosperm-specific maize B32 promoter (nucleotides 848 through 1259 ofGenBank accession number X70153, see also Hartings et al. (1990) PlantMol. Biol., 14:1031-1040, which is incorporated herein by reference),although other promoter elements could be used. This specific embodimentalso includes an aroA gene as an herbicidal selectable marker; otherselectable marker or reporter genes can be used. As shown in the lowerpart of FIG. 5B, the intron-embedded gene suppression element (“GSE”)can include any one or more gene suppression elements as described under“Gene Suppression Elements”.

FIG. 6A is a schematic map of a vector including an enhanced anti-senseconstruct as described in Example 5. The vector includes an aroA gene asan herbicidal selectable marker and a recombinant DNA construct forenhanced anti-sense gene suppression, consisting of a TUB-1 rootspecific promoter from Arabidopsis thaliana operably linked totranscribable DNA consisting of anti-sense oriented DNA of a nematodemajor sperm protein (msp) of a soybean cyst nematode, wherein afunctional polyadenylation site is absent in this transcribable DNA. Theplasmid also includes left T-DNA border (LB) and right T-DNA border (RB)elements. FIG. 6B is a schematic map of a recombinant DNA construct ofthe present invention as described in Example 5, which includes an aroAgene as an herbicidal selectable marker and a recombinant DNA constructof the present invention for gene suppression, including left T-DNAborder (LB) and right T-DNA border (RB) elements, and a TUB-1 rootspecific promoter from Arabidopsis thaliana operably linked to an intron(maize alcohol dehydrogenase intron, I-Zm-adh1) within which is embeddeda first transcribable heterologous DNA that includes an anti-sense DNAsegment that is anti-sense to the target gene, nematode major spermprotein of a soybean cyst nematode, wherein a functional polyadenylationsite is absent in this transcribable heterologous DNA.

FIG. 7A depicts a gene suppression element useful in a recombinant DNAconstruct of the invention, including intron-embedded tandem repeats forenhancing nuclear-localized gene silencing as described in Example 6.Such an element can be combined with at least one T-DNA border in theconstruct for Agrobacterium-mediated transformation of a plant cell. Theconstructs optionally include a gene expression element, which can beupstream (5′) or downstream (3′) of the intron. In a variation of thisembodiment (not shown), the intron-embedded tandem repeats are located3′ to the terminator. FIG. 7B shows another vector useful fornuclear-localized gene silencing by tandem repeats, wherein the vectorincludes tandem repeats transcribed from constructs lacking a functionalterminator. In a variation of this embodiment (not shown), the tandemrepeats are located 3′ to the terminator. FIG. 7C shows yet anothervector useful for nuclear-localized gene silencing by tandem repeats,wherein the vector includes tandem repeats under transcriptional controlof two opposing promoters.

FIG. 8A schematically depicts non-limiting recombinant DNA constructs ofthe invention as described in Example 8. For use inAgrobacterium-mediated transformation of plant cells, at least one T-DNAborder is generally included in each construct (not shown). Theseconstructs include a promoter element (“pro”), an intron flanked on oneor on both sides by non-protein-coding DNA, an optional terminatorelement (“ter”), at least one first gene suppression element (“GSE” or“GSE1”) for suppressing at least one first target gene, and canoptionally include at least one second gene suppression element (“GSE2”)for suppressing at least one second target gene, at least one geneexpression element (“GEE”) for expressing at least one gene of interest,or both. In embodiments containing a gene expression element, the geneexpression element can be located adjacent to (outside of) the intron.In one variation of this embodiment (not shown), the gene suppressionelement (embedded in an intron flanked on one or on both sides bynon-protein-coding DNA) is located 3′ to the terminator. In otherconstructs of the invention (not shown), a gene suppression element (notintron-embedded) is located 3′ to the terminator (see Example 22). FIG.8B schematically depicts examples of recombinant DNA constructs distinctfrom those of the present invention. These constructs can contain a genesuppression element that is located adjacent to an intron or between twodiscrete introns (that is to say, not embedded within a single intron),or can include a gene expression element including a gene suppressionelement embedded within an intron which is flanked on both sides byprotein-coding DNA (e. g., protein-coding exons that make up a geneexpression element).

FIG. 9 depicts various non-limiting examples of gene suppressionelements and transcribable exogenous DNAs useful in the recombinant DNAconstructs of the invention. Where drawn as a single strand (FIGS. 9Athrough 9E), these are conventionally depicted in 5′ to 3′ (left toright) transcriptional direction, where the arrows indicate anti-sensesequence (arrowhead pointing to the left), or sense sequence (arrowheadpointing to the right). Where drawn as double-stranded (anti-parallel)transcripts (FIGS. 9F and 9G), the 5′ and 3′ transcriptionaldirectionality is as shown. Solid lines, dashed lines, and dotted linesindicate sequences that target different target genes.

These gene suppression elements and transcribable exogenous DNAs caninclude: DNA that includes at least one anti-sense DNA segment that isanti-sense to at least one segment of the at least one first targetgene, or DNA that includes multiple copies of at least one anti-senseDNA segment that is anti-sense to at least one segment of the at leastone first target gene (FIG. 9A); DNA that includes at least one senseDNA segment that is at least one segment of the at least one firsttarget gene, or DNA that includes multiple copies of at least one senseDNA segment that is at least one segment of the at least one firsttarget gene (FIG. 9B); DNA that transcribes to RNA for suppressing theat least one first target gene by forming double-stranded RNA andincludes at least one anti-sense DNA segment that is anti-sense to atleast one segment of the at least one target gene and at least one senseDNA segment that is at least one segment of the at least one firsttarget gene (FIG. 9C); DNA that transcribes to RNA for suppressing theat least one first target gene by forming a single double-stranded RNAand includes multiple serial anti-sense DNA segments that are anti-senseto at least one segment of the at least one first target gene andmultiple serial sense DNA segments that are at least one segment of theat least one first target gene (FIG. 9D); DNA that transcribes to RNAfor suppressing the at least one first target gene by forming multipledouble strands of RNA and includes multiple anti-sense DNA segments thatare anti-sense to at least one segment of the at least one first targetgene and multiple sense DNA segments that are at least one segment ofthe at least one first target gene, and wherein said multiple anti-senseDNA segments and the multiple sense DNA segments are arranged in aseries of inverted repeats (FIG. 9E); and DNA that includes nucleotidesderived from a miRNA (see also FIG. 5B), or DNA that includesnucleotides of a siRNA (FIG. 9F). FIG. 9F depicts various non-limitingarrangements of double-stranded RNA (dsRNA) that can be transcribed fromembodiments of the gene suppression elements and transcribable exogenousDNAs useful in the recombinant DNA constructs of the invention. Whensuch dsRNA is formed, it can suppress one or more target genes, and canform a single double-stranded RNA or multiple double strands of RNA, ora single dsRNA “stem” or multiple “stems”. Where multiple dsRNA “stems”are formed, they can be arranged in “hammerheads” or “cloverleaf”arrangements. Spacer DNA is optional and can include sequence thattranscribes to an RNA (e. g., a large loop of antisense sequence of thetarget gene or an aptamer) that assumes a secondary structure orthree-dimensional configuration that confers on the transcript a desiredcharacteristic, such as increased stability, increased half-life invivo, or cell or tissue specificity.

FIG. 10A depicts a non-limiting gene suppression element (“GSE”) usefulin recombinant DNA constructs of the invention, as described in Example10. FIG. 10B depicts a representation of the type of RNA double hairpinmolecule that it would be expected to produce. In this example,orientations of the sequences are anti-sense followed by sense forsequence 1, then sense followed by anti-sense for sequence 2 (FIG. 10A).Analogous recombinant DNA constructs could be designed to provide RNAmolecules containing more than 2 double-stranded “stems”, as shown inFIG. 10C, which depicts an RNA molecule containing 3 “stems”.

FIG. 11 depicts fold-back structures of maize and soy MIR sequences, asdescribed in detail in Example 14. Nucleotides corresponding to themature miRNA are indicated by bold font, Watson-Crick base-pairing by avertical line, and base-pairing mismatches by a dot.

FIG. 12 depicts fold-back structures of maize and soy MIR sequences, asdescribed in detail in Example 15. Nucleotides corresponding to themature miRNA are indicated by bold font, Watson-Crick base-pairing by avertical line, and base-pairing mismatches by a dot.

FIG. 13 depicts a miR166 consensus fold-back structure (Griffiths-Jones(2004) Nucleic Acids Res., 32, Database Issue, D109-D111, which isincorporated by reference herein) with the nucleotides corresponding tothe mature miRNA indicated by the shaded nucleotides, as described inExample 16.

FIG. 14 depicts expression levels of the indicated mature miRNAs invarious tissues from maize, as described in detail in Example 17.

FIG. 15 depicts a non-limiting example of transcribable DNA sequenceincluding an exogenous miRNA recognition site, chloroplast-targetedTIC809 with a miRNA162 recognition site (in bold text) located in the 3′untranslated region (SEQ ID NO. 220), as described in detail in Example18. The translated amino acid sequence is also shown.

FIG. 16 depicts a non-limiting example of transcribable DNA sequenceincluding an exogenous miRNA recognition site, non-chloroplast-targetedTIC809 with a miRNA164 recognition site (in bold text) located in the 3′untranslated region (SEQ ID NO. 221), as described in detail in Example18. The translated amino acid sequence is also shown.

FIG. 17 depicts the strong and specific endosperm expression of themiR167g microRNA (SEQ ID NO. 4) cloned from maize endosperm, asdescribed in detail in Example 19. Northern blots of RNA from maize(LH59) tissues probed with an end-labeled mature miR167 22-mer LNA probespecific for SEQ ID NO. 4 (FIG. 17A) or with a ˜400 bp miR167ggene-specific probe (FIG. 17B). Transcription profiling of maize tissuescorroborated the Northern blot results (FIG. 17C); the transcriptcorresponding to miR167g was abundantly and specifically expressed inendosperm tissue (abundances are categorized as follows: >5000, highabundance, 97^(th) percentile; 700-5000, moderate abundance, 20^(th)percentile; 400-700, average abundance; 200-400, low abundance; <200,not detected). Selected abbreviations: “DAP” or “DA”, days afterpollination; “WK”, whole kernel, “endo”, endosperm.

FIG. 18 depicts a partial annotation map, including locations of themiR167a and miR167g genes and mature miRNAs, and promoter elements (e.g., TATA boxes), of the genomic cluster within which was identified themiR167g promoter sequences as described in detail in Example 19.Abbreviations: “PBF”, prolamin box binding factor; “ARF”auxin-responsive (auxin binding) factor; “NIT2”, activator ofnitrogen-related genes; “LYS14”, element that binds to UASLYS, anupstream activating element conferring Lys14- and adipatesemialdehyde-dependent activation and apparent repression; “GLN3”,element that binds the nitrogen upstream activation sequence ofglutamine synthetase.

FIG. 19 depicts Northern blots from a transient expression assay inNicotiana benthamiana. Small RNA blots were hybridized to probesspecific for the mature miRNAs predicted to be processed from miR164e(SEQ ID NO. 228) (FIG. 19A) and from an miRNA engineered to targetColorado potato beetle vacuolar ATPase (SEQ ID NO. 229) (FIG. 19B), asdescribed in detail in Example 20. Results show that the predictedmature miRNAs were processed efficiently in vivo.

FIG. 20 depicts results described in detail in Example 21. FIG. 20Adepicts the fold-back structure of SEQ ID NO. 236, the predicted miRNAprecursor for SEQ ID NO. 234; the mature miRNA is located at bases106-126, the corresponding miRNA* at bases 156-175, and another abundantmiRNA was also found to be located at bases 100-120 in the stem of thefold-back structure. “Count” refers to the number of occurrences of asmall RNA in the filtered set of 381,633 putative miRNA sequences thatwas analyzed. FIG. 20B depicts a transcription profile in soy tissuesfor the miRNA precursor SEQ ID NO. 236. FIG. 20C depicts a transcriptionprofile in soy tissues for a predicted target, polyphenol oxidase (SEQID NO. 250) for the mature miRNA (SEQ ID NO. 234).

FIG. 21 depicts results described in detail in Example 21. FIG. 21Adepicts the fold-back structure of SEQ ID NO. 239, the predicted miRNAprecursor for SEQ ID NO. 237; the mature miRNA is located at bases163-183, and the miRNA* at bases 18-63. “Count” refers to the number ofoccurrences of a small RNA in the filtered set of 381,633 putative miRNAsequences that was analyzed. FIG. 21B depicts a transcription profile insoy tissues for a predicted target, polyphenol oxidase (SEQ ID NO. 251)for the mature miRNA (SEQ ID NO. 237).

FIG. 22 depicts results described in detail in Example 21. FIG. 22Adepicts the fold-back structure of SEQ ID NO. 242, the predicted miRNAprecursor for SEQ ID NO. 240; the mature miRNA is located at bases87-107, and the miRNA* at bases 150-169. “Count” refers to the number ofoccurrences of a small RNA in the filtered set of 381,633 putative miRNAsequences that was analyzed.

FIG. 23 depicts results described in detail in Example 21. FIG. 23Adepicts the fold-back structure of SEQ ID NO. 245, the predicted miRNAprecursor for SEQ ID NO. 243; the mature miRNA is located at bases61-81, and the miRNA* at bases 109-129. “Count” refers to the number ofoccurrences of a small RNA in the filtered set of 381,633 putative miRNAsequences that was analyzed.

FIG. 24 depicts results described in detail in Example 21. FIG. 24A(top) depicts the fold-back structure of SEQ ID NO. 248, one of thepredicted miRNA precursors for SEQ ID NO. 246; the mature miRNA islocated at bases 157-178, and the miRNA* at bases 72-93. FIG. 24A(bottom) depicts the fold-back structure of SEQ ID NO. 249, anotherpredicted miRNA precursors for SEQ ID NO. 246; the mature miRNA islocated at bases 123-144, and the miRNA* at bases 58-79. “Count” refersto the number of occurrences of a small RNA in the filtered set of381,633 putative miRNA sequences that was analyzed. FIG. 24B (top)depicts a transcription profile in soy tissues for the miRNA precursorSEQ ID NO. 248. FIG. 24B (bottom) depicts a transcription profile in soytissues for the miRNA precursor SEQ ID NO. 249.

FIG. 25 depicts various embodiments of recombinant DNA constructsincluding a gene suppression element 3′ to a terminator, as described indetail in Example 22.

FIG. 26 depicts constructs and results described in detail in Example22. FIG. 26A depicts a recombinant DNA construct (pMON100552) forsuppressing a target gene (luciferase), containing a gene suppressionelement 3′ to a terminator. FIG. 26A depicts a control construct(pMON100553). Y-axis values are given as the logarithm of the ratio oflogarithm of the ratio of firefly luciferase to Renilla luciferase,“log(Fluc/Rluc)”; error bars are 95% confidence intervals.

FIG. 27 schematically depicts non-limiting embodiments of therecombinant DNA useful in making transgenic plants of the invention. Thetranscribable DNA includes DNA that transcribes to at least one RNAaptamer domain, and can further include DNA that transcribes to an RNAregulatory domain (which can act “in cis” or “in trans”). Usefulpromoters include any promoter capable of transcribing the transcribableDNA in a transgenic plant of the invention, e. g., a pol II promoter ora pol III promoter. Various embodiments can include introns,double-stranded RNA-forming regions, and/or microRNA recognition sites.Some embodiments can further include one or more separate geneexpression elements or gene suppression elements (shown here as a geneof interest, “GOI”, which can be positioned upstream or downstream ofthe transcribable DNA).

FIG. 28 depicts non-limiting embodiments of recombinant DNA useful inmaking transgenic plants of the invention, as described in Example 24.Abbreviations: “TS”, target sequence; “Ter”, terminator; a geneexpression element represented by a non-limiting gene of interest“dapA”, cordapA; “TSs_(up)”, a gene suppression element. FIG. 24Fdepicts one mechanism for an “on” riboswitch acting in cis. “RB”, rightT-DNA border element; “LB”, left T-DNA border element.

FIG. 29, top panel, depicts different systems of controlling expressionof a target sequence (in this non-limiting example, of green fluorescentprotein, “GFP”) as described in Example 28. The bottom panel depicts anon-limiting example of a riboswitch autoinduced by its own ligand(lysine), as described in Example 28.

FIG. 30 depicts a non-limiting example of a riboswitch in a binaryvector useful in making a transgenic plant of the invention, asdescribed in Example 29. “RB”, right T-DNA border element; “LB”, leftT-DNA border element; “Nos Ter”, Nos terminator.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Generally, the nomenclature usedand the manufacture or laboratory procedures described below are wellknown and commonly employed in the art. Conventional methods are usedfor these procedures, such as those provided in the art and variousgeneral references. Unless otherwise stated, nucleic acid sequences inthe text of this specification are given, when read from left to right,in the 5′ to 3′ direction. Where a term is provided in the singular, theinventors also contemplate aspects of the invention described by theplural of that term. The nomenclature used and the laboratory proceduresdescribed below are those well known and commonly employed in the art.Where there are discrepancies in terms and definitions used inreferences that are incorporated by reference, the terms used in thisapplication shall have the definitions given. Other technical terms usedhave their ordinary meaning in the art that they are used, asexemplified by a variety of technical dictionaries. The inventors do notintend to be limited to a mechanism or mode of action. Reference theretois provided for illustrative purposes only.

I. Selective Expression of a Target Sequence in Transgenic Plant Cells,Plants, and Seeds

The present invention provides a transgenic plant cell having in itsgenome recombinant DNA including transcribable DNA including DNA thattranscribes to an RNA aptamer capable of binding to a ligand. In someembodiments of the invention, for example, in transgenic plant cellsmade transgenic by Agrobacterium-mediated transformation, therecombinant DNA further includes at least one T-DNA border. In manyembodiments, the transcribable DNA further includes DNA that transcribesto regulatory RNA capable of regulating expression of a target sequence,wherein the regulation of the target sequence is dependent on theconformation of the regulatory RNA, and the conformation of theregulatory RNA is allosterically affected by the binding state of theRNA aptamer.

Further provided by the invention is a transgenic plant including aregenerated plant prepared from a transgenic plant cell having in itsgenome recombinant DNA including transcribable DNA including DNA thattranscribes to an RNA aptamer capable of binding to a ligand, or aprogeny plant (which may be a hybrid progeny plant) of the regeneratedplant. Such transgenic plants may be plants of any developmental stage,including seed, and include transgenic plants grown from such seed. Alsoclaimed are plant tissues regenerated from the transgenic plant cell ofthe invention.

In preferred embodiments, the transgenic plant cell or plant having inits genome recombinant DNA including transcribable DNA including DNAthat transcribes to an RNA aptamer capable of binding to a ligand has atleast one altered trait, relative to a plant lacking the recombinantDNA, as described in detail under the heading “Making and UsingTransgenic Plant Cells and Plants”. In these embodiments, the alteredtrait is typically obtained by providing the ligand to at least somecells or tissues of the transgenic plant. In one preferred embodiment,the altered trait is provided by contacting the transgenic plant with anexogenous ligand that binds to the aptamer. In some of theseembodiments, the exogenous ligand is physically applied to the plant (e.g., a synthetic or natural ligand applied to the plant as a foliar sprayor root solution), or applied (e. g., as a coating or soak) totransgenic seed of the transgenic plant. For example, the altered traitmay be obtained by contacting the transgenic plant with an herbicide (e.g., glyphosate or dicamba) that binds to an aptamer specific for theherbicide, thus turning “on” or “off” the regulatory RNA. In otherembodiments, the ligand is an exogenous ligand produced by or found in apest or pathogen of the transgenic plant, or a ligand (e. g., anallelochemical) produced by adjacent plants of the same or differentspecies as the transgenic plant. In another preferred embodiment, thealtered trait is obtained through the binding of an endogenous ligand tothe aptamer. In such embodiments, the ligand is endogenous to thetransgenic plant, e. g., a ligand produced constitutively, or in aspecific cell or tissue, or under biotic or abiotic stress, or at aparticular developmental or seasonal time. In a non-limiting example,the altered trait is obtained during a period of stress (biotic orabiotic), wherein a ligand, such as a stress-responsive molecule orhormone (e. g., salicylic acid, jasmonic acid, ethylene, glutathione,ascorbate, auxins, cytokinins), is endogenously produced by thetransgenic plant, and binds to an aptamer specific for thestress-responsive molecule. In yet another example, the altered traitmay be obtained in response to a pest or pathogen of the transgenicplant, wherein the aptamer is specific for a ligand produced by theplant in response to the pest or pathogen.

Transcribable DNA: The transcribable DNA includes DNA that transcribesto an RNA aptamer capable of binding to a ligand. By “transcribable” ismeant that the DNA is capable of being transcribed to RNA. Thus, inpreferred embodiments, the recombinant DNA further includes a promoteroperably linked to the transcribable DNA. Promoters of use in theinvention are preferably promoters functional in a plant cells, asdescribed under the heading “Promoter Elements”. Suitable promoters canbe constitutive or non-constitutive promoters. In various embodiments,the promoter element can include a promoter selected from the groupconsisting of a constitutive promoter, a spatially specific promoter, atemporally specific promoter, a developmentally specific promoter, andan inducible promoter. -In one embodiment of the invention, the promoteris a pol II promoter. In another embodiment, the promoter is a pol IIIpromoter (see, for example, Eckstein (2005) Trends Biochem. Sci.,30:445-452).

In many preferred embodiments, the transcribable DNA further includesDNA that transcribes to regulatory RNA capable of regulating expressionof a target sequence, wherein the regulation is dependent on theconformation of the regulatory RNA, and the conformation of theregulatory RNA is allosterically affected by the binding state of theRNA aptamer, that is to say, the conformation of the regulatory RNA isallosterically influenced by the conformation of the RNA aptamer, whichin turn is determined by whether the RNA aptamer is occupied orunoccupied by the specific ligand.

In some embodiments, the transcribable DNA is optionally flanked on oneor both sides by a ribozyme (e. g., a self-cleaving ribozyme or ahairpin ribozyme) (see, e. g., FIG. 27A). See, for example, Esteban etal. (1997) J. Biol. Chem., 272:13629-13639, which describes the effectsof conformation on hairpin ribozyme kinetics and provides guidelines forhairpin ribozyme sequence modification, and Najafi-Shoushtari et al.(2004) Nucleic Acids Res., 32:3212-3219, which describesconformationally controlled hairpin ribozymes. In other embodiments, thetranscribable DNA is optionally embedded within a spliceable intron(see, e. g., FIG. 27C). Introns suitable for use in the invention arepreferably introns that are spliceable in planta; plant-sourced intronsare especially preferred. Non-limiting examples of especially preferredplant introns include a rice actin 1 intron (I-Os-Act1), a maize heatshock protein intron (I-Zm-hsp70), and a maize alcohol dehydrogenaseintron (I-Zm-adh1). Embodiments where the transcribable DNA is flankedby intron splicing sites can further include additional sequence toallow cleavage of the transcript, e. g., DNA that transcribes to RNAincluding at least one microRNA recognition site or DNA that transcribesto RNA capable of forming double-stranded RNA (dsRNA) (see, e. g., FIG.27D). In other embodiments, the transcribable DNA includes DNA thattranscribes to RNA sequence that can be processed in an RNAi pathway (i.e., to produce small interfering RNAs or microRNAs, see, for example,Xie et al. (2004) PLoS Biol., 2:642-652; Bartel (2004) Cell,116:281-297; Murchison and Hannon (2004) Curr. Opin. Cell Biol.,16:223-229; and Dugas and Bartel (2004) Curr. Opin. Plant Biol.,7:512-520, which are incorporated by reference ). In non-limitingexamples, the transcribable DNA is optionally flanked by DNA thattranscribes to RNA including at least one microRNA recognition site(see, e. g., FIG. 27E). In these embodiments, the miRNA recognition siteis preferably a miRNA recognition site recognized by a miRNA endogenousto the plant in which transcription occurs. In a non-limiting example,the transcribable DNA is flanked on both sides by a miRNA recognitionsite that is recognized by a mature miRNA that is expressed in aninducible or a spatially or temporally specific manner. In yet otherembodiments, the transcribable DNA is optionally flanked on one or bothsides by DNA that transcribes to RNA capable of forming double-strandedRNA (dsRNA) (see, e. g., FIG. 27E), for example, by forming an invertedrepeat where the transcribable DNA is located in the middle “spacer” or“loop” region, or by forming separate dsRNA regions on one or both sidesof the transcribable DNA, which may be processed to small interferingRNAs or to mature microRNAs. In certain embodiments, the transcribableDNA can further include at least one gene expression (or suppression)element for the expression of any gene or genes or interest (includingcoding or non-coding sequence), as described under the heading “GeneExpression Elements” (see, e. g., FIG. 27C and FIG. 1D, where a geneexpression element is represented by a gene of interest, “GOI”, and FIG.28C and FIG. 28D, where a gene expression element is represented by aspecific gene of interest, cordapA, “dapA”, and FIG. 28E, where a genesuppression element is represented by “TS_(sup)”).

RNA Aptamers: Nucleic acid aptamers are nucleic acid molecules that bindto a ligand through binding mechanism that is not primarily based onWatson-Crick base-pairing (in contrast, for example, to the base-pairingthat occurs between complementary, anti-parallel nucleic acid strands toform a double-stranded nucleic acid structure). See, for example,Ellington and Szostak (1990) Nature, 346:818-822. A nucleic acid aptamergenerally includes a primary nucleotide sequence that allows the aptamerto form a secondary structure (e. g., by forming stem-loop structures)that allows the aptamer to bind to its ligand. Binding of the aptamer toits ligand is preferably specific, allowing the aptamer to distinguishbetween two or more molecules that are structurally similar (see, forexample, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343).Aptamers useful in the invention can, however, be monovalent (binding asingle ligand) or multivalent (binding more than one individual ligand,e. g., binding one unit of two or more different ligands). See, forexample, Di Giusto and King (2004) J. Biol. Chem., 279:46483-46489,describing the design and construction of multivalent, circular DNAaptamers, which is incorporated by reference.

Aptamers useful in the invention can include DNA, RNA, nucleic acidanalogues (e. g., peptide nucleic acids), locked nucleic acids,chemically modified nucleic acids, or combinations thereof. See, forexample, Schmidt et al. (2004) Nucleic Acids Res., 32:5757-5765, whodescribe locked nucleic acid aptamers. In one preferred embodiment ofthe invention, the aptamer is an RNA aptamer. In a particularlypreferred embodiment, the aptamer is produced by transcription inplanta. Examples of aptamers can be found, for example, in the publicAptamer Database, available on line at aptamer.icmb.utexas.edu (Lee etal. (2004) Nucleic Acids Res., 32(1):D95-100).

Aptamers can be designed for a given ligand by various procedures knownin the art, including in vitro selection or directed evolutiontechniques. See, for example, “SELEX” (“systematic evolution of ligandsby exponential enrichment”), as described in Tuerk and Gold (1990)Science, 249:505-510, Ellington and Szostak (1990) Nature, 346:818-822,Ellington and Szostak (1992) Nature, 355:850-852, selection ofbifunctional RNA aptamers by chimeric SELEX, as described by Burke andWillis (1998), RNA, 4:1165-1175, selection using ligands bound tomagnetic particles as described by Murphy et al. (2003) Nucleic AcidsRes., 31:e110, an automated SELEX technique described by Eulberg et al.(2005) Nucleic Acids Res., 33(4):e45, and a SELEX-type technique forobtaining aptamers raised against recombinant molecules expressed oncell surfaces, as described by Ohuchi et al. (2005) Nucleic AcidSymposium Series, 49:351-352 Selection can begin with a random pool ofRNAs, from a partially structured pool of RNAs (see, for example, Davisand Szostak (2002) Proc. Natl. Acad. Sci. USA, 99: 11616-11621), or froma pool of degenerate RNAs (see, for example, Geiger et al. (1996)Nucleic Acids Res., 24: 1029-1036). Secondary structure models, folding,and hybridization behavior for a given RNA sequence can be predictedusing algorithms, e. g., as described by Zuker (2003) Nucleic AcidsRes., 31: 3406-3415. Thus, aptamers for a given ligand can be designedde novo using suitable selection. One non-limiting example of aptamerdesign and selection is described in detail in Weill et al. (2004)Nucleic Acids Res., 32:5045-5058, which describes isolation of variousATP-binding aptamers and secondary selection of aptamers that bindcordycepin (3′ deoxyadenosine). Another non-limiting example of aptamerdesign is given in Huang and Szostak (2003) RNA, 9:1456-1463, whichdescribes the in vitro evolution of novel aptamers with newspecificities and new secondary structures from a starting aptamer. Allcitations in this paragraph are specifically incorporated by reference.

Ligands useful in the invention can include amino acids or theirbiosynthetic or catabolic intermediates, peptides, proteins,glycoproteins, lipoproteins, carbohydrates, fatty acids and otherlipids, steroids, terpenoids, hormones, nucleic acids, aromatics,alkaloids, natural products or synthetic compounds (e. g., dyes,pharmaceuticals, antibiotics, herbicides), inorganic ions, and metals,in short, any molecule (or part of a molecule) that can be recognizedand be bound by a nucleic acid secondary structure by a mechanism notprimarily based on Watson-Crick base pairing. In this way, therecognition and binding of ligand and aptamer is analogous to that ofantigen and antibody, or of biological effector and receptor. Ligandscan include single molecules (or part of a molecule), or a combinationof two or more molecules (or parts of a molecule), and can include oneor more macromolecular complexes (e. g., polymers, lipid bilayers,liposomes, cellular membranes or other cellular structures, or cellsurfaces). See, for example, Plummer et al. (2005) Nucleic Acids Res.,33:5602-5610, which describes selection of aptamers that bind to acomposite small molecule-protein surface; Zhuang et al. (2002) J. Biol.Chem., 277:13863-13872, which describes the association of insectmid-gut receptor proteins with lipid rafts, which affects the binding ofBacillus thuringiensis insecticidal endotoxins; and Homann and Goringer(1 999) Nucleic Acids Res., 27:2006-2014, which describes aptamers thatbind to live trypanosomes; these citations are incorporated byreference.

Non-limiting examples of specific ligands include vitamins such ascoenzyme B₁₂ and thiamine pyrophosphate, flavin mononucleotide, guanine,adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A,lysine, tyrosine, dopamine, glucosamine-6-phosphate, caffeine,theophylline, antibiotics such as chloramphenicol and neomycin,herbicides such as glyphosate and dicamba, proteins including viral orphage coat proteins and invertebrate epidermal or digestive tractsurface proteins, and RNAs including viral RNA, transfer-RNAs (t-RNAs),ribosomal RNA (rRNA), and RNA polymerases such as RNA-dependent RNApolymerase (RdRP). One class of RNA aptamers useful in the invention are“thermoswitches” that do not bind a ligand but are thermally responsive,that is to say, the aptamer's conformation is determined by temperature.See, for example, Box 3 in Mandal and Breaker (2004) Nature Rev. Mol.Cell Biol., 5:451-463, which is incorporated by reference.

An aptamer can be described by its binding state, that is, whether theaptamer is bound (or unbound) to its respective ligand. The binding site(or three-dimensional binding domain or domains) of an aptamer can bedescribed as occupied or unoccupied by the ligand. Similarly, apopulation of a given aptamer can be described by the fraction of thepopulation that is bound or unbound to the ligand. The affinity of anaptamer for its ligand can be described in terms of the rate ofassociation (binding) of the aptamer with the ligand and the rate ofdissociation of the ligand from the aptamer, e. g., by the equilibriumassociation constant (K) or by its reciprocal, the affinity constant(K_(a)) as is well known in the art. These rates can be determined bymethods similar to those commonly used for determining binding kineticsof ligands and receptors or antigens and antibodies, such as, but notlimited to, equilibrium assays, competition assays, surface plasmonresonance, and predictive models. The affinity of an aptamer for itsligand can be selected, e. g., during in vitro evolution of the aptamer,or further modified by changes to the aptamer's primary sequence, wheresuch changes can be guided by calculations of binding energy or byalgorithms, e. g., as described by Zuker (2003) Nucleic Acids Res.,31:3406-3415 or Bayer and Smolke (2005) Nature Biotechnol., 23:337-343.

The binding state of an aptamer preferably at least partially determinesthe secondary structure (e. g., the formation of double-stranded orsingle stranded regions) and the three-dimensional conformation of theaptamer. In embodiments where the transcribable DNA further includes DNAthat transcribes to regulatory RNA capable of regulating expression of atarget sequence, the binding state of the aptamer allosterically affectsthe conformation of the regulatory RNA and thus the ability of theregulatory RNA to regulate expression of the target sequence.

In one preferred embodiments, the aptamer (transcribed RNA) is flankedby DNA that transcribes to RNA capable of forming double-stranded RNA(dsRNA) (FIG. 27E). In some of these embodiments, the dsRNA is processedby an RNAi (siRNA or miRNA) mechanism, whereby the aptamer is cleavedfrom the rest of the transcript. In other, particularly preferredembodiments, the two transcribed RNA regions flanking the aptamer format least partially double-stranded RNA “stem” between themselves,wherein the aptamer serves as a “spacer” or “loop” in a stem-loopstructure; such an arrangement is expected to enhance the stability orhalf-life of the transcript in a manner analogous to that observed forDNA (see, for example, Di Giusto and King (2004) J. Biol. Chem.,279:46483-46489, which is incorporated by reference). Transgenic plantshaving in their genome DNA that transcribes to such aptamers havingenhanced stability are particularly desirable, e. g., where the aptamerfunctions to inhibit or kill a pathogen or pest of the transgenic plant.

Target Sequence: The regulatory RNA is capable of regulating expressionof a target sequence, wherein the regulation of the target sequence isdependent on the conformation of the regulatory RNA, and theconformation of the regulatory RNA is allosterically affected by thebinding state of the RNA aptamer. Any target sequence may be chosen,including one or more target sequences selected from a gene native tothe transgenic plant of the invention, a transgene in the transgenicplant, and a gene native to a pest or pathogen of the transgenic plant.The target sequence can include a sequence that expresses a gene ofinterest (e. g., an RNA encoding a protein), or a sequence thatsuppresses a gene of interest (e. g., an RNA that is processed to ansiRNA or miRNA that in turn suppresses the gene of interest).

The regulatory RNA can regulate the transcription and/or translation ofany target nucleic acid sequence or sequences of interest. In someembodiments, the recombinant DNA further includes a second generegulatory element for regulating (i. e., suppressing or expressing) atleast one second target sequence that is in addition to the targetsequence regulated by the regulatory RNA. Whether a first targetsequence or a second target sequence, the target sequence can include asingle sequence or part of a single sequence that is targetted forregulation, or can include, for example, multiple consecutive segmentsof a target sequence, multiple non-consecutive segments of a targetsequence, multiple alleles of a target sequence, or multiple targetsequences from one or more species.

The target sequence can be translatable (coding) sequence, or can benon-coding sequence (such as non-coding regulatory sequence), or both.The target sequence can include at least one eukaryotic target sequence,at least one non-eukaryotic target sequence, or both. A target sequencecan include any sequence from any species (including, but not limitedto, non-eukaryotes such as bacteria, and viruses; fungi; plants,including monocots and dicots, such as crop plants, ornamental plants,and non-domesticated or wild plants; invertebrates such as arthropods,annelids, nematodes, and molluscs; and vertebrates such as amphibians,fish, birds, domestic or wild mammals, and even humans. Suitable targetsequences are further described as “target genes” under the heading“Target Genes”.

Non-limiting examples of a target sequence include non-translatable(non-coding) sequence, such as, but not limited to, 5′ untranslatedregions, promoters, enhancers, or other non-coding transcriptionalregions, 3′ untranslated regions, terminators, and introns. Targetsequences can also include genes encoding microRNAs, small interferingRNAs, RNA components of ribosomes or ribozymes, small nucleolar RNAs,and other non-coding RNAs (see, for example, non-coding RNA sequencesprovided publicly at rfam.wustl.edu; Erdmann et al. (2001) Nucleic AcidsRes., 29:189-193; Gottesman (2005) Trends Genet., 21:399-404;Griffiths-Jones et al. (2005) Nucleic Acids Res., 33:121-124, which areincorporated by reference ). One specific example of a target sequenceincludes a microRNA recognition site (that is, the site on an RNA strandto which a mature miRNA binds and induces cleavage). Another specificexample of a target sequence includes a microRNA precursor sequence,that is, the primary transcript encoding a microRNA, or the RNAintermediates processed from this primary transcript (e. g., anuclear-limited pri-miRNA or a pre-miRNA which can be exported from thenucleus into the cytoplasm). See, for example, Lee et al. (2002) EMBOJournal, 21:4663-4670; Reinhart et al. (2002) Genes & Dev.,16:161611626; Lund et al. (2004) Science, 303:95-98; and Millar andWaterhouse (2005) Funct. Integr Genomics, 5:129-135, which areincorporated by reference. Target microRNA precursor DNA sequences canbe native to the transgenic plant of the invention, or can be native toa pest or pathogen of the transgenic plant. Target sequences can alsoinclude translatable (coding) sequence for genes encoding transcriptionfactors and genes encoding enzymes involved in the biosynthesis orcatabolism of molecules of interest (such as, but not limited to, aminoacids, fatty acids and other lipids, sugars and other carbohydrates,biological polymers, and secondary metabolites including alkaloids,terpenoids, polyketides, non-ribosomal peptides, and secondarymetabolites of mixed biosynthetic origin). A target sequence can be anative gene targetted for expression control (e. g., suppression), withor without concurrent expression (or suppression) of an exogenoustransgene, for example, by including a gene expression (or suppression)element in the same or in a separate recombinant DNA construct. Forexample, it can be desirable to replace a native gene with an exogenoustransgene homologue.

One preferred embodiment of the invention provides transgenic plantcells (or transgenic plants, progeny plants, or seeds derived from thetransgenic plant cells) having in their genome a recombinant DNAincluding transcribable DNA including DNA that transcribes to an RNAaptamer capable of binding to a ligand, for suppressing a plant pest orpathogen (e. g., viruses, bacteria, fungi, and invertebrates such asinsects, nematodes, and molluscs).

Examples of such embodiments include transgenic plant cells (ortransgenic plants, progeny plants, or seeds derived from the transgenicplant cells) having in their genome a recombinant DNA includingtranscribable DNA including DNA that transcribes to one or more RNAaptamers that bind to one or more ligands involved in a pest orpathogen's ability to recognize, invade, or feed on a plant, or in thepest or pathogen's ability to recruit additional individuals of itsspecies, or in the pest or pathogen's ability to grow, metamorphose, orreproduce. Non-limiting examples of ligands suitable for this approachinclude the insect mid-gut brush border receptor proteins that arerecognized by Bacillus thuringiensis insecticidal endotoxins. See, forexample, Knight et al. (1995) J. Biol. Chem., 270:17765-17770, and Gillet al. (1995) J. Biol. Chem., 270:27277-27282, which describe theisolation, identification, and cloning of examples of such receptorproteins; Gomez et al. (2001) J. Biol. Chem., 276:28906-28912, andDaniel et al. (2002) Appl. Env. Microbiol., 68:2106-2112, which describetechniques for identifying binding epitopes of such receptor proteinsand for studying their binding affinities; Jurat-Fuentes and Adang(2001) Appl. Env. Microbiol., 67:323-329, and Jurat-Fuentes et al.(2001), Appl. Env. Microbiol., 67:872-879, which describeendotoxin-receptor binding assays involving either membrane blots orsurface plasmon resonance measured binding of brush border membranevesicles to endotoxin; all of these are incorporated by reference. Otherexamples of suitable ligands to which RNA aptamers of the invention bindinclude steroid receptors, such as estrogen receptors, androgenreceptors, retinoid receptors, and ecdysone receptors (see, for example,Saez et al. (2000) Proc. Natl. Acad. Sci. USA, 97:14512-14517. Whereligands are receptor molecules or receptor complexes, RNA aptamers ofthe invention can optionally act as antagonists or as agonists.

One aspect of the invention provides transgenic plants wherein thetarget sequence is selected to provide resistance to a plant pest orpathogen, for example, resistance to a nematode such as soybean cystnematode or root knot nematode or to a pest insect. Thus, targetsequences (i. e., “target genes”) of interest can also includeendogenous genes of plant pests and pathogens as described in detailunder “Target Genes”. Pests and pathogens of interest includeinvertebrates (including nematodes, molluscs, and insects), fungi,bacteria, mollicute, and viruses, as described in detail under “TargetGenes”. Thus, a target sequence need not be endogenous to the plant inwhich the recombinant DNA is transcribed. It is envisioned thatrecombinant DNA of the invention can be transcribed in a plant and usedto control expression of a target sequence endogenous to a pathogen orpest that may infest the plant.

Regulatory RNA: In many embodiments, the transcribable DNA furtherincludes DNA that transcribes to regulatory RNA capable of regulatingexpression of a target sequence, wherein the regulation of the targetsequence is dependent on the conformation of the regulatory RNA, and theconformation of the regulatory RNA is allosterically affected by thebinding state of the RNA aptamer. Such combinations of an aptamer with aregulator RNA domain are commonly known as riboswitches. The regulatoryRNA is typically downstream of the aptamer but the two domains mayoverlap; see, e. g., Najafi-Shoushtari and Famulok (2005) RNA,11:1514-1520, which is incorporated by reference and describes a hairpinribozyme that includes an aptamer domain and is competitively regulatedby flavin mononucleotide and an oligonucleotide complementary to theaptamer domain. In some embodiments, the regulatory RNA is operablylinked to the target sequence, and acts “in cis”. In other embodiments,the regulatory RNA is not operably linked to the target sequence, andacts “in trans”.

In riboswitch embodiments including an aptamer and a regulatory RNA, theriboswitch regulates expression of the target sequence by any suitablemechanism. One non-limiting mechanism is transcriptional regulation bythe ligand-dependent formation of an intrinsic terminator stem (anextended stem-loop structure typically followed by a run of 6 or more Uresidues) that causes RNA polymerase to abort transcription, e. g.,before a complete mRNA is formed. In “off” riboswitches, in the absenceof sufficient ligand, the unbound aptamer domain permits formation of an“antiterminator stem”, which prevents formation of the intrinsicterminator stem and thus allows transcription to proceed; thus, thedefault state of the riboswitch is “on” (i. e., transcription normallyproceeds) and the ligand must be added to turn the riboswitch off. In“on” riboswitches that use this mechanism, the aptamer domain must be inthe bound (ligand-occupied) conformation to permit formation of the“antiterminator stem” and allow transcription. Another mechanism istranslation regulation, where ligand binding causes structural changesin full-length mRNAs and thereby permits (or prevents) ribosomes frombinding to the ribosomal binding site (RBS); the formation of an“anti-anti-RBS” stem and an “anti-RBS” stem is also mutually exclusive.In “on” riboswitches that use this mechanism, absence of the ligandallows formation of an anti-anti-RBS, and thus a structurallyunencumbered RBS to which the ribosome can bind. A combination of bothtranscriptional and translational regulation is also possible. For adetailed discussion of regulation mechanisms, see Mandal and Breaker(2004) Nature Rev. Mol. Cell Biol., 5:451-463, which is incorporated byreference.

In some embodiments, the regulatory RNA includes a ribozyme, e. g., aself-cleaving ribozyme, a hammerhead ribozyme, or a hairpin ribozyme.Certain embodiments of the regulatory RNA include RNA sequence that iscomplementary or substantially complementary to the target sequence. Onenon-limiting example is where the regulatory RNA includes an anti-sensesegment that is complementary or substantially complementary to thetarget sequence. See, for example, Bayer and Smolke (2005) NatureBiotechnol., 23:337-343, where the regulatory RNA includes both ananti-sense segment complementary to the target sequence, and a sensesegment complementary to the anti-sense segment, wherein the anti-sensesegment and sense segment are capable of hybridizing to each other toform an intramolecular double-stranded RNA.

In embodiments where regulation of a target sequence involvesWatson-Crick base-pairing of the regulatory RNA to the target sequence(e. g., in trans-acting embodiments, see, e. g., Bayer and Smolke (2005)Nature Biotechnol., 23:337-343), the target sequence of interest can bemore specifically targetted by designing the regulatory RNA to includeregions substantially non-identical to a non-target sequence sequence.Non-target sequences can include any gene for which the expression ispreferably not modified, either in a plant transcribing the recombinantDNA construct or in organisms that may come into contact with RNAtranscribed from the recombinant DNA construct. A non-target sequencecan include any sequence from any species (including, but not limitedto, non-eukaryotes such as bacteria, and viruses; fungi; plants,including monocots and dicots, such as crop plants, ornamental plants,and non-domesticated or wild plants; invertebrates such as arthropods,annelids, nematodes, and molluscs; and vertebrates such as amphibians,fish, birds, domestic or wild mammals, and even humans).

In one embodiment of the invention, the target sequence is a geneendogenous to a given species, such as a given plant (such as, but notlimited to, agriculturally or commercially important plants, includingmonocots and dicots), and the non-target sequence can be, for example, agene of a non-target species, such as another plant species or a gene ofa virus, fungus, bacterium, invertebrate, or vertebrate, even a human.One non-limiting example is where it is desirable to design either theaptamer, or the regulatory RNA, or both, in order to modify theexpression of a target sequence that is a gene endogenous to a singlespecies (e. g., Western corn rootworm, Diabrotica virgifera virgiferaLeConte) but to not modify the expression of a non-target sequence suchas genes from related, even closely related, species (e. g., Northerncorn rootworm, Diabrotica barberi Smith and Lawrence, or Southern cornrootworm, Diabrotica undecimpunctata).

In other embodiments (e. g., where it is desirable to modify theexpression of a target sequence across multiple species), it may bedesirable to design the aptamer, or the regulatory RNA, or both, tomodify the expression of a target sequence common to the multiplespecies in which the expression of the target sequence is to bemodified. Thus, the aptamer, or the regulatory RNA, or both, can beselected to be specific for one taxon (e. g., specific to a genus,family, or even a larger taxon such as a phylum, e. g., arthropoda) butnot for other taxa (for example, plants or vertebrates or mammals). Inone non-limiting example of this embodiment, a regulatory RNA can beselected so as to target pathogenic fungi (e. g., a Fusarium spp.) butnot target any gene sequence from beneficial fungi (e. g., beneficialsoil mycorrhizal fungi).

In another non-limiting example of this embodiment, the aptamer, or theregulatory RNA, or both, to regulate gene expression in corn rootwormcan be selected to be specific to all members of the genus Diabrotica.For example, a regulatory RNA including a Diabrotica-targettedsuppression element (e. g., anti-sense RNA, double-stranded RNA,microRNA, or tandem RNA repeats) can be selected so as to not target anygene sequence from beneficial coleopterans (for example, predatorycoccinellid beetles, commonly known as ladybugs or ladybirds) or otherbeneficial insect species.

The required degree of specificity of a regulatory RNA that includes agene suppression element (e. g., anti-sense RNA, double-stranded RNA,microRNA, or tandem RNA repeats) for suppression of a target sequencedepends on various factors. For example, where the gene suppressionelement includes double-stranded RNA (dsRNA), factors can include thesize of the smaller dsRNA fragments that are expected to be produced bythe action of Dicer, and the relative importance of decreasing thedsRNA's potential to suppress non-target sequences. For example, wherethe dsRNA fragments are expected to be 21 base pairs in size, oneparticularly preferred embodiment can be to include in the regulatoryRNA a sequence capable of forming dsRNA and encoding regionssubstantially non-identical to a non-target sequence, such as regionswithin which every contiguous fragment including at least 21 nucleotidesmatches fewer than 21 (e. g., fewer than 21, or fewer than 20, or fewerthan 19, or fewer than 18, or fewer than 17) out of 21 contiguousnucleotides of a non-target sequence. In another embodiment, regionssubstantially non-identical to a non-target sequence include regionswithin which every contiguous fragment including at least 19 nucleotidesmatches fewer than 19 (e. g., fewer than 19, or fewer than 18, or fewerthan 17, or fewer than 16) out of 19 contiguous nucleotides of anon-target sequence.

In some embodiments, it may be desirable to design the aptamer, theregulatory RNA, or both, to include regions predicted to not generateundesirable polypeptides, for example, by screening the aptamer, theregulatory RNA, or both, for sequences that may encode known undesirablepolypeptides or close homologues of these. Undesirable polypeptidesinclude, but are not limited to, polypeptides homologous to knownallergenic polypeptides and polypeptides homologous to known polypeptidetoxins. Publicly available sequences encoding such undesirablepotentially allergenic peptides are available, for example, the FoodAllergy Research and Resource Program (FARRP) allergen database(available at allergenonline.com) or the Biotechnology Information forFood Safety Databases (available at www.iit.edu/˜sgendel/fa.htm) (seealso, for example, Gendel (1998) Adv. Food Nutr. Res., 42:63-92, whichis incorporated by reference). Undesirable sequences can also include,for example, those polypeptide sequences annotated as known toxins or aspotential or known allergens and contained in publicly availabledatabases such as GenBank, EMBL, SwissProt, and others, which aresearchable by the Entrez system (www.ncbi.nih.gov/Entrez). Non-limitingexamples of undesirable, potentially allergenic peptide sequencesinclude glycinin from soybean, oleosin and agglutinin from peanut,glutenins from wheat, casein, lactalbumin, and lactoglobulin from bovinemilk, and tropomyosin from various shellfish (allergenonline.com).Non-limiting examples of undesirable, potentially toxic peptides includetetanus toxin tetA from Clostridium tetani, diarrheal toxins fromStaphylococcus aureus, and venoms such as conotoxins from Conus spp. andneurotoxins from arthropods and reptiles (www.ncbi.nih.gov/Entrez).

In one non-limiting example, a proposed aptamer, regulatory RNA, orboth, can be screened to eliminate those transcribable sequencesencoding polypeptides with perfect homology to a known allergen or toxinover 8 contiguous amino acids, or with at least 35% identity over atleast 80 amino acids; such screens can be performed on any and allpossible reading frames in both directions, on potential open readingframes that begin with ATG, or on all possible reading frames,regardless of whether they start with an ATG or not. When a “hit” ormatch is made, that is, when a sequence that encodes a potentialpolypeptide with perfect homology to a known allergen or toxin over 8contiguous amino acids (or at least about 35% identity over at leastabout 80 amino acids), is identified, the DNA sequences corresponding tothe hit can be avoided, eliminated, or modified when selecting sequencesto be used in the aptamer, the regulatory RNA, or both.

Avoiding, elimination of, or modification of, an undesired sequence canbe achieved by any of a number of methods known to those skilled in theart. In some cases, the result can be novel sequences that are believedto not exist naturally. For example, avoiding certain sequences can beaccomplished by joining together “clean” sequences into novel chimericsequences to be used in a gene suppression element.

Where the regulatory RNA includes double-stranded RNA (dsRNA) forsilencing a target gene, applicants recognize that in somedsRNA-mediated gene silencing, it is possible for imperfectly matchingdsRNA sequences to be effective at gene silencing. For example, it hasbeen shown that mismatches near the center of a miRNA complementary sitehas stronger effects on the miRNA's gene silencing than do more distallylocated mismatches. See, for example, FIG. 4 in Mallory et al. (2004)EMBO J., 23:3356-3364, which is incorporated by reference. In anotherexample, it has been reported that, both the position of a mismatchedbase pair and the identity of the nucleotides forming the mismatchinfluence the ability of a given siRNA to silence a target sequence, andthat adenine-cytosine mismatches, in addition to the G:U wobble basepair, were well tolerated (see Du et al. (2005) Nucleic Acids Res.,33:1671-1677, which is incorporated by reference). Thus, a regulatoryRNA that includes double-stranded RNA need not always have 100% sequenceidentity with the intended target sequence, but generally wouldpreferably have substantial sequence identity with the intended targetsequence, such as about 95%, about 90%, about 85%, or about 80% sequenceidentity with the intended target sequence. One skilled in the art wouldbe capable of judging the importance given to screening for regionspredicted to be more highly specific to the first target sequence orpredicted to not generate undesirable polypeptides, relative to theimportance given to other criteria, such as, but not limited to, thepercent sequence identity with the intended first target sequence or thepredicted gene silencing efficiency of a given sequence. For example, itmay be desirable for a given regulatory RNA that includesdouble-stranded RNA for gene silencing to be active across severalspecies, and therefore one skilled in the art can determine that it ismore important to include in the regulatory RNA regions specific to theseveral species of interest, but less important to screen for regionspredicted to have higher gene silencing efficiency or for regionspredicted to generate undesirable polypeptides.

In many embodiments, the transgenic plant cell has in its genomerecombinant DNA including transcribable DNA including (a) DNA thattranscribes to an RNA aptamer capable of binding to a ligand, and (b)DNA that transcribes to regulatory RNA capable of regulating expressionof a target sequence, wherein the regulation is dependent on theconformation of the regulatory RNA, and the conformation of saidregulatory RNA is allosterically affected by the binding state of saidRNA aptamer. In these embodiments, binding of the aptamer to its ligandresults in a specific change in the expression of the target sequence,which may be an increase or a decrease in expression, depending on thedesign of the recombinant DNA.

In one embodiment, binding of the ligand to the RNA aptamer results inan increase of expression of the target sequence relative to expressionin the absence of the binding. In another embodiment, binding of theligand to the RNA aptamer results in a decrease of expression of thetarget sequence relative to expression in the absence of the binding.

Some embodiments are characterized by “autoinducibility”. In one suchembodiment, binding of the ligand to the RNA aptamer results in anincrease of expression of the target sequence relative to expression inthe absence of the binding, wherein the increase of expression resultsin a level of the ligand sufficient to maintain the increase ofexpression. In another embodiment, binding of the ligand to the RNAaptamer results in a decrease of expression of the target sequencerelative to expression in the absence of the binding, the decrease ofexpression resulting in a level of the ligand sufficient to maintain theincrease of expression.

Thus, another aspect of the invention is a method of modifyingexpression of a gene of interest in a plant cell, including transcribingin a transgenic plant cell of the invention, or a plant, progeny plant,or seed or other plant tissue derived from such a transgenic plant cell,recombinant or heterologous DNA that transcribes to (a) an RNA aptamercapable of binding to a ligand, and (b) regulatory RNA capable ofregulating expression of a target sequence, wherein the regulation isdependent on the conformation of the regulatory RNA, and wherein theconformation of the regulatory RNA is allosterically affected by thebinding state of the RNA aptamer, whereby expression of the gene ofinterest is modified relative to its expression in the absence oftranscription of the recombinant DNA construct.

Method of Reducing Invertebrate Pest Damage to a Plant: The presentinvention also provides a method of reducing damage to a plant by a pestor pathogen of the plant, including transcribing in the plant arecombinant DNA construct including transcribable DNA including DNA thattranscribes to an RNA aptamer capable of binding to a ligand, whereinthe ligand includes at least part of a molecule endogenous to the pestor pathogen, and whereby binding of the RNA aptamer to the ligandreduces damage to the plant by the pest or pathogen, relative to damagein the absence of transcription of the recombinant DNA construct. Theligand can include at least part of any molecule that is part of apest's anatomy (e. g., a coat or surface protein or macromolecularstructure), or is produced or secreted by the pest or pathogen (e. g.,an enzyme secreted by a pathogen in invasion of a plant cell)

In particularly preferred embodiments, the pest or pathogen is aninvertebrate pest of the transgenic plant, and the ligand includes atleast part of a molecule of the digestive tract lining of theinvertebrate pest, e. g., insect mid-gut brush border receptor proteinsthat are recognized by Bacillus thuringiensis insecticidal endotoxins(see discussion above under the heading “RNA Aptamers”). Invertebratepests of interest are listed above under the heading “Target Sequences”.

The invention also contemplates and claims an analogous method forimproving resistance in a transgenic plant to bacterial, fungal, orviral pathogens. The method reduces damage to a transgenic plant by abacterial, fungal, or viral pathogen of the plant, including the step oftranscribing in the plant a recombinant DNA construct includingtranscribable DNA including DNA that transcribes to an RNA aptamercapable of binding to a ligand, wherein the ligand includes at leastpart of a molecule endogenous to the bacterial, fungal, or viralpathogen, and whereby binding of the RNA aptamer to the ligand reducesdamage to the plant by the bacterial, fungal, or viral pathogen,relative to damage in the absence of transcription of the recombinantDNA construct. Bacterial, fungal, and viral pathogens of interest areprovided under the heading “Target Genes”.

Recombinant DNA Constructs: The present invention further provides arecombinant DNA construct including: (a) transcribable DNA including DNAthat transcribes to an RNA aptamer capable of binding to a ligand; and(b) DNA sequence that transcribes to double-stranded RNA flanking saidtranscribable DNA. In some embodiments, the recombinant DNA constructfurther includes DNA that transcribes to regulatory RNA capable ofregulating expression of a target sequence, wherein the regulation isdependent on the conformation of the regulatory RNA, and theconformation of the regulatory RNA is allosterically affected by thebinding state of the RNA aptamer. The transcribable DNA is DNA that iscapable of being transcribed in a eukaryotic cell, preferably an animalcell or a plant cell.

The double-stranded RNA (dsRNA) is preferably RNA that is capable ofbeing processed through an RNAi pathway (i. e., to produce smallinterfering RNAs or microRNAs, see, for example, Xie et al. (2004) PLoSBiol., 2:642-652; Bartel (2004) Cell, 116:281-297; Murchison and Hannon(2004) Curr. Opin. Cell Biol., 16:223-229; and Dugas and Bartel (2004)Curr. Opin. Plant Biol., 7:512-520, which are incorporated byreference). The RNAi pathway can be that found in animals or that foundin plants. See, e. g., Lee et al. (2002) EMBO Journal, 21:4663-4670;Reinhart et al. (2002) Genes & Dev., 16:161611626; Lund et al. (2004)Science, 303:95-98; and Millar and Waterhouse (2005) Funct. IntegrGenomics, 5:129-135, which are incorporated by reference. Whereas inanimals both miRNAs and siRNAs are believed to result from activity ofthe same DICER enzyme, in plants miRNAs and siRNAs are formed bydistinct DICER-like (DCL) enzymes, and in Arabidopsis a nuclear DCLenzyme is believed to be required for mature miRNA formation (Xie et al.(2004) PLoS Biol., 2:642-652, which is incorporated by reference).

In non-limiting examples, the transcribable DNA is optionally flanked onone or both sides by DNA that transcribes to RNA capable of formingdouble-stranded RNA (dsRNA) (for example, by forming an inverted repeatwhere the transcribable DNA is located in the middle “spacer” region, orby forming separate dsRNA regions on one or both sides of thetranscribable DNA, which may be processed to small interfering RNAs, tomicroRNA precursors such as pre-miRNAs, or to mature microRNAs). In yetother embodiments, the transcribable DNA is optionally flanked by DNAthat transcribes to RNA including at least one microRNA recognitionsite. In these embodiments, the miRNA recognition site is preferably amiRNA recognition site recognized by a miRNA endogenous to the plant inwhich transcription occurs. In a non-limiting example, the transcribableDNA is flanked on both sides by a miRNA recognition site that isrecognized by a mature miRNA that is expressed in an inducible or aspatially or temporally specific manner. The transcribable DNA canfurther include at least one gene expression element.

The invention further provides a transgenic eukaryotic cell including inits genome a recombinant DNA construct including: (a) transcribable DNAincluding DNA that transcribes to an RNA aptamer capable of binding to aligand; and (b) DNA sequence that transcribes to double-stranded RNAflanking said transcribable DNA. Such cells may be animal cells or plantcells. Also provided is a transgenic plant having in its genome arecombinant DNA construct including: (a) transcribable DNA including DNAthat transcribes to an RNA aptamer capable of binding to a ligand; and(b) DNA sequence that transcribes to double-stranded RNA flanking saidtranscribable DNA Methods for preparing and using the recombinant DNAconstructs, and for making transgenic cells and transgenic plants, aredescribed under the headings “Making and Using Recombinant DNAConstructs” and “Making and Using Transgenic Plant Cells and TransgenicPlants”.

II. Recombinant DNA Constructs Containing Introns and Gene SuppressionElements

The present invention provides a recombinant DNA construct for planttransformation including a promoter operably linked to a first genesuppression element for suppressing at least one first target gene,wherein said first gene suppression element is embedded in an intronflanked on one or on both sides by non-protein-coding DNA. In someembodiments, the recombinant DNA construct consists entirely ofnon-protein-coding DNA (e. g., a promoter, a gene suppression elementthat is embedded in an intron and that transcribes to a non-coding RNA,and an optional terminator). Thus, the invention includes the use of anintron to deliver a gene suppression element in the absence of anyprotein-coding exons.

In some embodiments, the intron is located adjacent to at least oneelement selected from the group consisting of the promoter and aterminator, that is to say, directly contiguous (or essentiallydirectly, with no substantial intervening sequence) with the promoter orwith a terminator or with both. In one specific embodiment, the intronis directly (or essentially directly) 3′ to the promoter. The intron canalso optionally be directly (or essentially directly) 5′ to aterminator, if a terminator is present in the recombinant DNA construct.Where the intron is adjacent to a terminator element, any interveningsequence preferably does not include a self-splicing ribozyme. In onepreferred embodiment, the intron containing the gene suppression elementis flanked directly (on the 5′ end) by the promoter element, and (on the3′ end) by the terminator element if one is present.

The inventors have unexpectedly found that transcription can continuedownstream of a terminator at least sufficiently to allow transcriptionof a gene suppression element located 3′ to the terminator (downstreamof a polyadenylation sequence). Thus another aspect of the invention isa recombinant DNA construct including a promoter, a terminator,transcribable sequence (which can include coding or non-coding sequenceor both, and can include, e. g., a gene expression element, a genesuppression element, an aptamer, or a riboswitch) between the promoterand the terminator, and at least one gene suppression element that is 3′to the terminator. In various embodiments, at least one gene suppressionelement (such as any one or more of those described under “GeneSuppression Elements”), whether embedded in an intron or not, is locateddownstream of a terminator and sufficiently proximate to the terminatorto permit transcription of the gene suppression element. In a specificembodiment, the intron is located downstream of a terminator andsufficiently proximate to the terminator to permit transcription of theintron. In one preferred but non-limiting embodiment, the intron isdirectly (or essentially directly) 3′ to a terminator. Introns canaffect the expression of adjacent sequences (e. g., depending on theintron's splicing efficiency), and thus one advantage of placing a genesuppression element (or intron containing a gene suppression element) 3′to a terminator includes allowing expression of a sequence between thepromoter and the terminator, wherein the expression is not influenced byin the manner that it may be if the gene suppression element (or introncontaining a gene suppression element) was also located between thepromoter and the terminator. Another advantage includes the likelihoodthat a gene suppression element 3′ to a terminator will be processed asan aberrant transcript (e. g., converted to double-stranded RNA in anRNA-dependent RNA polymerase manner even in the absence of invertedrepeat sequences), which can increase the efficiency of gene suppression(see Examples 1, 2, and 3, which illustrate that lack of sequencesnecessary for polyadenylation enhanced the efficiency of a genesuppression element). Yet another advantage is that this approachreduces the need for multiple promoter elements, especially useful whenstacking multiple genetic constructs to be expressed in a single cell.

The recombinant DNA construct contains one or more first genesuppression element for suppressing at least one first target gene andembedded in an intron flanked on one or on both sides bynon-protein-coding DNA. Suitable gene suppression elements are describedunder the heading “Gene Suppression Elements”. Where the recombinant DNAconstruct contains more than one first gene suppression element, each ofthese first gene suppression elements can include one or more elementsas described herein. The first target gene can include a single gene orpart of a single gene that is targetted for suppression, or can include,for example, multiple consecutive segments of a first target gene,multiple non-consecutive segments of a first target gene, multiplealleles of a first target gene, or multiple first target genes from oneor more species. Suitable first target genes are described under theheading “Target Genes”.

Introns of use in the recombinant DNA construct are described under theheading “Introns”. The intron is located adjacent to at least oneelement selected from the group consisting of a promoter element and aterminator element, as described under the headings “Promoter Elements”and “Terminator Elements” respectively. Preferably, upon transcriptionof the recombinant DNA construct, the first gene suppression element isspliced out of the intron. In some embodiments, the recombinant DNAconstruct is designed so that the RNA transcribed from the first genesuppression element, when spliced out of the intron, lacks at least oneof a functional polyadenylation signal or a functional polyadenylationsite (or any other element that facilitates transport of a transcribedRNA into the cytoplasm), or lacks a 3′ untranslated region; theresulting transcribed RNA (and gene suppression by the transcribed RNA)is preferably localized in the nucleus. In other embodiments, therecombinant DNA construct is designed so that the RNA transcribed fromthe first gene suppression element, when spliced out of the intron, istransported out of the nucleus for gene suppression in the cytoplasm.

In various embodiments of the invention, the recombinant DNA constructsare optionally characterized by any one or more of the following. Therecombinant DNA construct can further include at least one of: (a) atleast one T-DNA border region, as described under “T-DNA Borders”; (b)spacer DNA, as described under “Spacer DNA”; (c) a gene expressionelement for expressing at least one gene of interest, wherein the geneexpression element is located adjacent to the intron; (d) a geneexpression element for expressing at least one gene of interest, whereinsaid gene expression element is located adjacent to said first genesuppression element and within said intron; and (e) a second genesuppression element for suppressing at least one second target gene,wherein the second gene suppression element is located outside of (e.g., adjacent to) the intron. These further aspects are described in moredetail below.

In some embodiments, the recombinant DNA construct further includes agene expression element for expressing at least one gene of interest,wherein the gene expression element is located adjacent to the intron.The gene of interest can include a single gene or multiple genes. Geneexpression elements are further described under the heading “GeneExpression Elements”.

In yet other embodiments, the recombinant DNA construct further includesa second gene suppression element for suppressing at least one secondtarget gene, wherein the second gene suppression element is locatedoutside of, e. g., adjacent to, the intron. The at least one secondtarget gene can include a single gene or part of a single gene that istargetted for suppression, or can include, for example, multipleconsecutive segments of a second target gene, multiple non-consecutivesegments of a second target gene, multiple alleles of a second targetgene, or multiple second target genes from one or more species. Suitablesecond target genes are described under the heading “Target Genes”.

Gene Suppression Elements: The gene suppression element can betranscribable DNA of any suitable length, and will generally include atleast about 19 to about 27 nucleotides (for example 19, 20, 21, 22, 23,or 24 nucleotides) for every target gene that the recombinant DNAconstruct is intended to suppress. In many embodiments the genesuppression element includes more than 23 nucleotides (for example, morethan about 30, about 50, about 100, about 200, about 300, about 500,about 1000, about 1500, about 2000, about 3000, about 4000, or about5000 nucleotides) for every target gene that the recombinant DNAconstruct is intended to suppress.

Suitable gene suppression elements useful in the recombinant DNAconstructs of the invention include at least one element (and, in someembodiments, multiple elements) selected from the group consisting of:

-   (a) DNA that includes at least one anti-sense DNA segment that is    anti-sense to at least one segment of the at least one first target    gene;-   (b) DNA that includes multiple copies of at least one anti-sense DNA    segment that is anti-sense to at least one segment of the at least    one first target gene;-   (c) DNA that includes at least one sense DNA segment that is at    least one segment of the at least one first target gene;-   (d) DNA that includes multiple copies of at least one sense DNA    segment that is at least one segment of the at least one first    target gene;-   (e) DNA that transcribes to RNA for suppressing the at least one    first target gene by forming double-stranded RNA and includes at    least one anti-sense DNA segment that is anti-sense to at least one    segment of the at least one target gene and at least one sense DNA    segment that is at least one segment of the at least one first    target gene;-   (f) DNA that transcribes to RNA for suppressing the at least one    first target gene by forming a single double-stranded RNA and    includes multiple serial anti-sense DNA segments that are anti-sense    to at least one segment of the at least one first target gene and    multiple serial sense DNA segments that are at least one segment of    the at least one first target gene;-   (g) DNA that transcribes to RNA for suppressing the at least one    first target gene by forming multiple double strands of RNA and    includes multiple anti-sense DNA segments that are anti-sense to at    least one segment of the at least one first target gene and multiple    sense DNA segments that are at least one segment of the at least one    first target gene, and wherein said multiple anti-sense DNA segments    and the multiple sense DNA segments are arranged in a series of    inverted repeats;-   (h) DNA that includes nucleotides derived from a miRNA, preferably a    plant miRNA;-   (i) DNA that includes nucleotides of a siRNA;-   (j) DNA that transcribes to an RNA aptamer capable of binding to a    ligand; and-   (k) DNA that transcribes to an RNA aptamer capable of binding to a    ligand, and DNA that transcribes to regulatory RNA capable of    regulating expression of the first target gene, wherein the    regulation is dependent on the conformation of the regulatory RNA,    and the conformation of the regulatory RNA is allosterically    affected by the binding state of the RNA aptamer.

Any of these gene suppression elements, whether transcribing to a singledouble-stranded RNA or to multiple double-stranded RNAs, can be designedto suppress more than one target gene, including, for example, more thanone allele of a target gene, multiple target genes (or multiple segmentsof at least one target gene) from a single species, or target genes fromdifferent species.

Anti-Sense DNA Segments: In one embodiment, the at least one anti-senseDNA segment that is anti-sense to at least one segment of the at leastone first target gene includes DNA sequence that is anti-sense orcomplementary to at least a segment of the at least one first targetgene, and can include multiple anti-sense DNA segments, that is,multiple copies of at least one anti-sense DNA segment that isanti-sense to at least one segment of the at least one first targetgene. Multiple anti-sense DNA segments can include DNA sequence that isanti-sense or complementary to multiple segments of the at least onefirst target gene, or to multiple copies of a segment of the at leastone first target gene, or to segments of multiple first target genes, orto any combination of these. Multiple anti-sense DNA segments can befused into a chimera, e. g., including DNA sequences that are anti-senseto multiple segments of one or more first target genes and fusedtogether.

The anti-sense DNA sequence that is anti-sense or complementary to (thatis, can form Watson-Crick base-pairs with) at least a segment of the atleast one first target gene has preferably at least about 80%, or atleast about 85%, or at least about 90%, or at least about 95%complementarity to at least a segment of the at least one first targetgene. In one preferred embodiment, the DNA sequence that is anti-senseor complementary to at least a segment of the at least one first targetgene has between about 95% to about 100% complementarity to at least asegment of the at least one first target gene. Where the at least oneanti-sense DNA segment includes multiple anti-sense DNA segments, thedegree of complementarity can be, but need not be, identical for all ofthe multiple anti-sense DNA segments.

Sense DNA Segments: In another embodiment, the at least one sense DNAsegment that is at least one segment of the at least one first targetgene includes DNA sequence that corresponds to (that is, has a sequencethat is identical or substantially identical to) at least a segment ofthe at least one first target gene, and can include multiple sense DNAsegments, that is, multiple copies of at least one sense DNA segmentthat corresponds to (that is, has the nucleotide sequence of) at leastone segment of the at least one first target gene. Multiple sense DNAsegments can include DNA sequence that is or that corresponds tomultiple segments of the at least one first target gene, or to multiplecopies of a segment of the at least one first target gene, or tosegments of multiple first target genes, or to any combination of these.Multiple sense DNA segments can be fused into a chimera, that is, caninclude DNA sequences corresponding to multiple segments of one or morefirst target genes and fused together.

The sense DNA sequence that corresponds to at least a segment of thetarget gene has preferably at least about 80%, or at least about 85%, orat least about 90%, or at least about 95% sequence identity to at leasta segment of the target gene. In one preferred embodiment, the DNAsequence that corresponds to at least a segment of the target gene hasbetween about 95% to about 100% sequence identity to at least a segmentof the target gene. Where the at least one sense DNA segment includesmultiple sense DNA segments, the degree of sequence identity can be, butneed not be, identical for all of the multiple sense DNA segments.

Multiple Copies: Where the gene suppression element includes multiplecopies of anti-sense or multiple copies of sense DNA sequence, thesemultiple copies can be arranged serially in tandem repeats. In someembodiments, these multiple copies can be arranged serially end-to-end,that is, in directly connected tandem repeats. In some embodiments,these multiple copies can be arranged serially in interrupted tandemrepeats, where one or more spacer DNA segment can be located adjacent toone or more of the multiple copies. Tandem repeats, whether directlyconnected or interrupted or a combination of both, can include multiplecopies of a single anti-sense or multiple copies of a single sense DNAsequence in a serial arrangement or can include multiple copies of morethan one anti-sense DNA sequence or of more than one sense DNA sequencein a serial arrangement.

Double-stranded RNA: In those embodiments wherein the gene suppressionelement includes either at least one anti-sense DNA segment that isanti-sense to at least one segment of the at least one target gene or atleast one sense DNA segment that is at least one segment of the at leastone target gene, RNA transcribed from either the at least one anti-senseor at least one sense DNA may become double-stranded by the action of anRNA-dependent RNA polymerase. See, for example, U.S. Pat. No. 5,283,184,which is incorporated by reference herein.

In yet other embodiments, the gene suppression element can include DNAthat transcribes to RNA for suppressing the at least one first targetgene by forming double-stranded RNA and includes at least one anti-senseDNA segment that is anti-sense to at least one segment of the at leastone target gene (as described above under the heading “Anti-sense DNASegments”) and at least one sense DNA segment that is at least onesegment of the at least one first target gene (as described above underthe heading “Sense DNA Segments”). Such a gene suppression element canfurther include spacer DNA segments. Each at least one anti-sense DNAsegment is complementary to at least part of a sense DNA segment inorder to permit formation of double-stranded RNA by intramolecularhybridization of the at least one anti-sense DNA segment and the atleast one sense DNA segment. Such complementarity between an anti-senseDNA segment and a sense DNA segment can be, but need not be, 100%complementarity; in some embodiments, this complementarity can bepreferably at least about 80%, or at least about 85%, or at least about90%, or at least about 95% complementarity.

The double-stranded RNA can be in the form of a single dsRNA “stem”(region of base-pairing between sense and anti-sense strands), or canhave multiple dsRNA “stems”. In one embodiment, the gene suppressionelement can include DNA that transcribes to RNA for suppressing the atleast one first target gene by forming essentially a singledouble-stranded RNA and includes multiple serial anti-sense DNA segmentsthat are anti-sense to at least one segment of the at least one firsttarget gene and multiple serial sense DNA segments that are at least onesegment of the at least one first target gene; the multiple serialanti-sense and multiple serial sense segments can form a singledouble-stranded RNA “stem” or multiple “stems” in a serial arrangement(with or without non-base paired spacer DNA separating the multiple“stems”). In another embodiment, the gene suppression element includesDNA that transcribes to RNA for suppressing the at least one firsttarget gene by forming multiple dsRNA “stems” of RNA and includesmultiple anti-sense DNA segments that are anti-sense to at least onesegment of the at least one first target gene and multiple sense DNAsegments that are at least one segment of the at least one first targetgene, and wherein said multiple anti-sense DNA segments and the multiplesense DNA segments are arranged in a series of dsRNA “stems” (such as,but not limited to “inverted repeats”). Such multiple dsRNA “stems” canfurther be arranged in series or clusters to form tandem invertedrepeats, or structures resembling “hammerhead” or “cloverleaf” shapes.Any of these gene suppression elements can further include spacer DNAsegments found within a dsRNA “stem” (for example, as a spacer betweenmultiple anti-sense or sense DNA segments or as a spacer between abase-pairing anti-sense DNA segment and a sense DNA segment) or outsideof a double-stranded RNA “stem” (for example, as a loop regionseparating a pair of inverted repeats). In cases where base-pairinganti-sense and sense DNA segment are of unequal length, the longersegment can act as a spacer. FIGS. 5B and 9 depict illustrations ofpossible embodiments of these gene suppression constructs.

miRNAs: In a further embodiment, the gene suppression element caninclude DNA that includes nucleotides derived from a miRNA (microRNA),that is, a DNA sequence that corresponds to a miRNA native to a virus ora eukaryote of interest (including plants and animals, especiallyinvertebrates), or a DNA sequence derived from such a native miRNA butmodified to include nucleotide sequences that do not correspond to thenative miRNA. While miRNAs have not to date been reported in fungi,fungal miRNAs, should they exist, are also suitable for use in theinvention. A particularly preferred embodiment includes a genesuppression element containing DNA that includes nucleotides derivedfrom a viral or plant miRNA.

In a non-limiting example, the nucleotides derived from a miRNA caninclude DNA that includes nucleotides corresponding to the loop regionof a native miRNA and nucleotides that are selected from a target genesequence. In another non-limiting example, the nucleotides derived froma miRNA can include DNA derived from a miRNA precursor sequence, such asa native pri-miRNA or pre-miRNA sequence, or nucleotides correspondingto the regions of a native miRNA and nucleotides that are selected froma target gene sequence number such that the overall structure (e. g.,the placement of mismatches in the stem structure of the pre-miRNA) ispreserved to permit the pre-miRNA to be processed into a mature miRNA.In yet another embodiment, the gene suppression element can include DNAthat includes nucleotides derived from a miRNA and capable of inducingor guiding in-phase cleavage of an endogenous transcript intotrans-acting siRNAs, as described by Allen et al. (2005) Cell,121:207-221, which is incorporated by reference in its entirety herein.Thus, the DNA that includes nucleotides derived from a miRNA can includesequence naturally occurring in a miRNA or a miRNA precursor molecule,synthetic sequence, or both.

siRNAs: In yet another embodiment, the gene suppression element caninclude DNA that includes nucleotides of a small interfering RNA(siRNA). The siRNA can be one or more native siRNAs (such as siRNAsisolated from a non-transgenic eukaryote or from a transgeniceukaryote), or can be one or more DNA sequences predicted to have siRNAactivity (such as by use of predictive tools known in the art, see, forexample, Reynolds et al. (2004) Nature Biotechnol., 22:326-330, which isincorporated by reference in its entirety herein). Multiple native orpredicted siRNA sequences can be joined in a chimeric siRNA sequence forgene suppression. Such a DNA that includes nucleotides of a siRNApreferably includes at least 19 nucleotides, and in some embodimentspreferably includes at least 21, at least 22, at least 23, or at least24 nucleotides. In other embodiments, the DNA that includes nucleotidesof a siRNA can contain substantially more than 21 nucleotides, forexample, more than about 50, about 100, about 300, about 500, about1000, about 3000, or about 5000 nucleotides or greater.

Introns: As used herein, “intron” or “intron sequence” generally meansnon-coding DNA sequence from a natural gene, which retains in therecombinant DNA constructs of this invention its native capability to beexcised from pre-mRNA transcripts, e. g., native intron sequences foundwith associated protein coding RNA regions, wherein the native intronsare spliced, allowing exons to be assembled into mature mRNAs before theRNA leaves the nucleus. Such an excisable intron has a 5′ splice siteand a 3′ splice site. Introns can be self-splicing or non-self-splicing(that is, requiring enzymes or a spliceosome for splicing to occur) andcan be selected for different splicing efficiency.

Introns suitable for use in constructs of the invention can be viralintrons (e. g., Yamada et al. (1994) Nucleic Acids Res., 22:2532-2537),eukaryotic introns (including animal, fungal, and plant introns),archeal or bacterial introns (e. g., Belfort et al. (1995) J.Bacteriol., 177:3897-3903), or any naturally occurring or artificial (e.g., Yoshimatsu and Nagawa (1989) Science, 244:1346-1348) DNA sequenceswith intron-like functionality in the plant in which the recombinant DNAconstruct of the invention is to be transcribed. While essentially anyintron can be used in the practice of this invention as a host forembedded DNA, particularly preferred are introns that are introns thatenhance expression in a plant or introns that are derived from a 5′untranslated leader sequence. Where a recombinant DNA construct of theinvention is used to transform a plant, plant-sourced introns can beespecially preferred. Examples of especially preferred plant intronsinclude a rice actin 1 intron (I-Os-Act1) (Wang et al. (1992) Mol. CellBiol., 12:3399-3406; McElroy et al. (1990) Plant Cell, 2:163-171), amaize heat shock protein intron (I-Zm-hsp70) (U.S. Pat. Nos. 5,593,874and 5,859,347), and a maize alcohol dehydrogenase intron (I-Zm-adh1)(Callis et al. (1987) Genes Dev., 1:1183-1200). Other examples ofintrons suitable for use in the invention include the tobacco mosaicvirus 5′ leader sequence or “omega” leader (Gallie and Walbot (1992)Nucleic Acids Res., 20:4631-4638), the Shrunken-1(Sh-1) intron (Vasil etal. (1989) Plant Physiol., 91:1575-1579), the maize sucrose synthaseintron (Clancy and Hannah (2002) Plant Physiol., 130:918-929), the heatshock protein 18 (hsp18) intron (Silva et al. (1987) J. Cell Biol.,105:245), and the 82 kilodalton heat shock protein (hsp82) intron(Semrau et al. (1989) J. Cell Biol., 109, p. 39A, and Mettler et al.(May 1990) N.A.T.O. Advanced Studies Institute on Molecular Biology,Elmer, Bavaria).

Promoter Elements: Where the recombinant DNA construct is to betranscribed in an animal cell, the promoter element is functional in ananimal. Where the recombinant DNA construct is to be transcribed in anplant cell, the promoter element is functional in a plant. Preferredpromoter elements include promoters that have promoter activity in aplant transformed with the recombinant DNA constructs of the invention.Suitable promoters can be constitutive or non-constitutive promoters. Invarious embodiments, the promoter element can include a promoterselected from the group consisting of a constitutive promoter, aspatially specific promoter, a temporally specific promoter, adevelopmentally specific promoter, and an inducible promoter.

Non-constitutive promoters suitable for use with the recombinant DNAconstructs of the invention include spatially specific promoters,temporally specific promoters, and inducible promoters. Wheretranscription of the construct is to occur in a plant cell, spatiallyspecific promoters can include organelle-, cell-, tissue-, ororgan-specific promoters functional in a plant (e. g., aplastid-specific, a root-specific, a pollen-specific, or a seed-specificpromoter for suppressing expression of the first target RNA in plastids,roots, pollen, or seeds, respectively). In many cases a seed-specific,embryo-specific, aleurone-specific, or endosperm-specific promoter isespecially useful. Where transcription of the construct is to occur inan animal cell, spatially specific promoters include promoters that haveenhanced activity in a particular animal cell or tissue (e. g., enhancedor specific promoter activity in nervous tissue, liver, muscle, eye,blood, marrow, breast, prostate, gonads, or other tissues). Temporallyspecific promoters can include promoters that tend to promote expressionduring certain developmental stages in an animal or plant's growth orreproductive cycle, or during different times of day or night, or atdifferent seasons in a year. Inducible promoters include promotersinduced by chemicals (e. g., exogenous or synthetic chemicals as well asendogenous pheromones and other signaling molecules) or by environmentalconditions such as, but not limited to, biotic or abiotic stress (e. g.,water deficit or drought, heat, cold, high or low nutrient or saltlevels, high or low light levels, or pest or pathogen infection). Anexpression-specific promoter can also include promoters that aregenerally constitutively expressed but at differing degrees or“strengths” of expression, including promoters commonly regarded as“strong promoters” or as “weak promoters”.

In one particularly preferred embodiment, the promoter element includesa promoter element functional in a plant transformed with a recombinantDNA construct of the invention. Non-limiting specific examples includean opaline synthase promoter isolated from T-DNA of Agrobacterium, and acauliflower mosaic virus 35S promoter, among others, as well as enhancedpromoter elements or chimeric promoter elements, e. g., an enhancedcauliflower mosaic virus (CaMV) 35S promoter linked to an enhancerelement (an intron from heat shock protein 70 of Zea mays). Manyexpression-specific promoters functional in plants and useful in themethod of the invention are known in the art. For example, U.S. Pat.Nos. 5,837,848; 6,437,217 and 6,426,446 disclose root specificpromoters; U.S. Pat. No. 6,433,252 discloses a maize L3 oleosinpromoter; U.S. Patent Application Publication 2004/0216189 discloses apromoter for a plant nuclear gene encoding a plastid-localized aldolase;U.S. Pat. No. 6,084,089 discloses cold-inducible promoters; U.S. Pat.No. 6,140,078 discloses salt inducible promoters; U.S. Pat. No.6,294,714 discloses light-inducible promoters; U.S. Pat. No. 6,252,138discloses pathogen-inducible promoters; and U.S. Patent ApplicationPublication 2004/0123347 A1 discloses water deficit-inducible promoters.All of the above-described patents and patent publications disclosingpromoters and their use, especially in recombinant DNA constructsfunctional in plants, are incorporated herein by reference.

The promoter element can include nucleic acid sequences that are notnaturally occurring promoters or promoter elements or homologues thereofbut that can regulate expression of a gene. Examples of such “geneindependent” regulatory sequences include naturally occurring orartificially designed RNA sequences that include a ligand-binding regionor aptamer and a regulatory region (which can be cis-acting). See, forexample, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer andSmolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004)Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005)Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature,419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal andBreaker (2004) Nature Struct. Mol. Biol., 11:29-35, all of which areincorporated by reference herein. Such “riboregulators” could beselected or designed for specific spatial or temporal specificity, forexample, to regulate translation of the exogenous gene only in thepresence (or absence) of a given concentration of the appropriateligand.

Terminator Elements: In some embodiments, the recombinant DNA constructincludes both a promoter element and a functional terminator element.Where it is functional, the terminator element includes a functionalpolyadenylation signal and polyadenylation site, allowing RNAtranscribed from the recombinant DNA construct to be polyadenylated andprocessed for transport into the cytoplasm.

In other embodiments, a functional terminator element is absent. In someembodiments where a functional terminator element is absent, at leastone of a functional polyadenylation signal and a functionalpolyadenylation site is absent. In other embodiments, a 3′ untranslatedregion is absent. In these cases, the recombinant DNA construct istranscribed as unpolyadenylated RNA and is preferably not transportedinto the cytoplasm.

T-DNA Borders: T-DNA borders refer to the DNA sequences or regions ofDNA that define the start and end of an Agrobacterium T-DNA (tumor DNA)and function in cis for transfer of T-DNA into a plant genome byAgrobacterium-mediated transformation (see, e. g., Hooykaas andSchilperoort (1992) Plant Mol. Biol., 19:15-38). In one preferredembodiment of the recombinant DNA construct of the invention, the intronin which is embedded the gene suppression element is located between apair of T-DNA borders, which can be a set of left and right T-DNAborders, a set of two left T-DNA borders, or a set of two right T-DNAborders. In another embodiment, the recombinant DNA construct includes asingle T-DNA border and an intron-embedded gene suppression element.

Spacer DNA: Spacer DNA segments can include virtually any DNA (such as,but not limited to, translatable DNA sequence encoding a gene ofinterest, translatable DNA sequence encoding a marker or reporter gene;transcribable DNA derived from an intron, which upon transcription canbe excised from the resulting transcribed RNA; transcribable DNAsequence encoding RNA that forms a structure such as a loop or stem oran aptamer capable of binding to a specific ligand; spliceable DNA suchas introns and self-splicing ribozymes; transcribable DNA encoding asequence for detection by nucleic acid hybridization, amplification, orsequencing; and a combination of these). Spacer DNA can be found, forexample, between parts of a gene suppression element, or betweendifferent gene suppression elements. In some embodiments, spacer DNA isitself sense or anti-sense sequence of the target gene. In somepreferred embodiments, the RNA transcribed from the spacer DNA (e. g., alarge loop of antisense sequence of the target gene or an aptamer)assumes a secondary structure or three-dimensional configuration thatconfers on the transcript a desired characteristic, such as increasedstability, increased half-life in vivo, or cell or tissue specificity.

Target Genes: The recombinant DNA construct can be designed to suppressany first target gene. In some embodiments, the construct furtherincludes a second gene suppression element for suppressing at least onesecond target gene, wherein the second gene suppression element islocated adjacent to the intron. Whether a first or a second target gene,the target gene can include a single gene or part of a single gene thatis targetted for suppression, or can include, e. g., multipleconsecutive segments of a target gene, multiple non-consecutive segmentsof a target gene, multiple alleles of a target gene, or multiple targetgenes from one or more species.

The target gene can be translatable (coding) sequence, or can benon-coding sequence (such as non-coding regulatory sequence), or both,and can include at least one gene selected from the group consisting ofa eukaryotic target gene, a non-eukaryotic target gene, a microRNAprecursor DNA sequence, and a microRNA promoter. The target gene can benative (endogenous) to the cell (e. g., a cell of a plant or animal) inwhich the recombinant DNA construct of the invention is transcribed, orcan be native to a pest or pathogen of the plant or animal in which theconstruct is transcribed. The target gene can be an exogenous gene, suchas a transgene in a plant.

The target gene can include a single gene or part of a single gene thatis targetted for suppression, or can include, for example, multipleconsecutive segments of a target gene, multiple non-consecutive segmentsof a target gene, multiple alleles of a target gene, or multiple targetgenes from one or more species. A target gene sequence can include anysequence from any species (including, but not limited to, non-eukaryotessuch as bacteria, and viruses; fungi; plants, including monocots anddicots, such as crop plants, ornamental plants, and non-domesticated orwild plants; invertebrates such as arthropods, annelids, nematodes, andmolluscs; and vertebrates such as amphibians, fish, birds, domestic orwild mammals, and even humans.

Non-limiting examples of a target gene include non-translatable(non-coding) sequence, such as, but not limited to, 5′ untranslatedregions, promoters, enhancers, or other non-coding transcriptionalregions, 3′ untranslated regions, terminators, and introns. Target genescan also include genes encoding microRNAs, small interfering RNAs, RNAcomponents of ribosomes or ribozymes, small nucleolar RNAs, and othernon-coding RNAs (see, for example, non-coding RNA sequences providedpublicly at rfam.wustl.edu; Erdmann et al. (2001) Nucleic Acids Res.,29:189-193; Gottesman (2005) Trends Genet., 21:399-404; Griffiths-Joneset al. (2005) Nucleic Acids Res., 33:121-124, which are incorporated byreference herein). One specific example of a target gene includes amicroRNA precursor DNA sequence, that is, the primary DNA transcriptencoding a microRNA, or the RNA intermediates processed from thisprimary transcript (e. g., a nuclear-limited pri-miRNA or a pre-miRNAwhich can be exported from the nucleus into the cytoplasm), or amicroRNA promoter. See, for example, Lee et al. (2002) EMBO Journal,21:4663-4670; Reinhart et al. (2002) Genes & Dev., 16:161611626; Lund etal. (2004) Science, 303:95-98; and Millar and Waterhouse (2005) Funct.Integr Genomics, 5:129-135, which are incorporated by reference herein.In one non-limiting embodiment, the target gene includes nucleotides ofa loop region of at least one target microRNA precursor. In plants,microRNA precursor molecules (e. g., primary miRNA transcripts) arebelieved to be largely processed in the nucleus, and thus recombinantDNA constructs of the invention that are transcribed tonon-polyadenylated suppression transcripts are expected to suppressthese and other nuclear-localized target genes in plants moreeffectively than conventional gene suppression constructs that resultin, e. g., double-stranded RNA molecules localized in the cytoplasm.Target microRNA precursor DNA sequences can be native to the transgenicplant in which the recombinant DNA construct of the invention istranscribed, or can be native to a pest or pathogen of the transgenicplant. Target genes can also include translatable (coding) sequence forgenes encoding transcription factors and genes encoding enzymes involvedin the biosynthesis or catabolism of molecules of interest (such as, butnot limited to, amino acids, fatty acids and other lipids, sugars andother carbohydrates, biological polymers, and secondary metabolitesincluding alkaloids, terpenoids, polyketides, non-ribosomal peptides,and secondary metabolites of mixed biosynthetic origin). A target genecan be a native gene targetted for suppression, with or withoutconcurrent expression of an exogenous transgene, for example, byincluding a gene expression element in the same or in a separaterecombinant DNA construct. For example, it can be desirable to replace anative gene with an exogenous transgene homologue.

It can be useful to provide transgenic plants having in their genome aDNA construct for suppressing a gene which is exogenous to the hostplant but endogenous to a plant pest or pathogen (e. g., viruses,bacteria, fungi, and invertebrates such as insects, nematodes, andmolluscs). Thus, one aspect of the invention provides recombinant DNAconstructs wherein the target gene is selected to provide resistance toa plant pest or pathogen, for example, resistance to a nematode such assoybean cyst nematode or root knot nematode or to a pest insect. Thus,target genes of interest can also include endogenous genes of plantpests and pathogens. Pest invertebrates include, but are not limited to,pest nematodes (e. g., cyst nematodes Heterodera spp. especially soybeancyst nematode Heterodera glycines, root knot nematodes Meloidogyne spp.,lance nematodes Hoplolaimus spp., stunt nematodes Tylenchorhynchus spp.,spiral nematodes Helicotylenchus spp., lesion nematodes Pratylenchusspp., ring nematodes Criconema spp., and foliar nematodes Aphelenchusspp. or Aphelenchoides spp.), pest molluscs (slugs and snails), and pestinsects (e. g., corn rootworms, Lygus spp., aphids, corn borers,cutworms, armyworms, leafhoppers, Japanese beetles, grasshoppers, andother pest coelepterans, dipterans, and lepidopterans). Plant pathogensof interest include fungi (e. g., the fungi that cause powdery mildew,rust, leaf spot and blight, damping-off, root rot, crown rot, cottonboll rot, stem canker, twig canker, vascular wilt, smut, or mold,including, but not limited to, Fusarium spp., Phakospora spp.,Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyricularia spp.,Alternaria spp., and Phytophthora spp.), bacteria (e. g., the bacteriathat cause leaf spotting, fireblight, crown gall, and bacterial wilt),mollicutes (e. g., the mycoplasmas that cause yellows disease andspiroplasmas such as Spiroplasma kunkelii, which causes corn stunt), andviruses (e. g., the viruses that cause mosaics, vein banding, flecking,spotting, or abnormal growth). See also G. N. Agrios, “Plant Pathology”(Fourth Edition), Academic Press, San Diego, 1997, 635 pp., which isincorporated by reference herein, for descriptions of fungi, bacteria,mollicutes (including mycoplasmas and spiroplasmas), viruses, nematodes,parasitic higher plants, and flagellate protozoans, all of which areplant pests or pathogens of interest. See also the continually updatedcompilation of plant pests and pathogens and the diseases caused by suchon the American Phytopathological Society's “Common Names of PlantDiseases”, compiled by the Committee on Standardization of Common Namesfor Plant Diseases of The American Phytopathological Society, 1978-2005,available online at www.apsnet.org/online/common/top.asp, which isincorporated by reference herein.

Non-limiting examples of fungal plant pathogens of particular interestinclude Phakospora pachirhizi (Asian soy rust), Puccinia sorghi (corncommon rust), Puccinia polysora (corn Southern rust), Fusarium oxysporumand other Fusarium spp., Alternaria spp., Penicillium spp., Pythiumaphanidermatum and other Pythium spp., Rhizoctonia solani, Exserohilumturcicum (Northern corn leaf blight), Bipolaris maydis (Southern cornleaf blight), Ustilago maydis (corn smut), Fusarium graminearum(Gibberella zeae), Fusarium verticilliodes (Gibberella moniliformis), F.proliferatum (G. fujikuroi var. intermedia), F. subglutinans (G.subglutinans), Diplodia maydis, Sporisorium holci-sorghi, Colletotrichumgraminicola, Setosphaeria turcica, Aureobasidium zeae, Phytophthorainfestans, Phytophthora sojae, Sclerotinia sclerotiorum, and thenumerous fungal species provided in Tables 4 and 5 of U.S. Pat. No.6,194,636, which is incorporated in its entirety by reference herein.

Non-limiting examples of bacterial pathogens include Pseudomonas avenae,Pseudomonas andropogonis, Erwinia stewartii, Pseudomonas syringae pv.syringae, and the numerous bacterial species listed in Table 3 of U.S.Pat. No. 6,194,636, which is incorporated in its entirety by referenceherein.

Non-limiting examples of viral plant pathogens of particular interestinclude maize dwarf mosaic virus (MDMV), sugarcane mosaic virus (SCMV,formerly MDMV strain B), wheat streak mosaic virus (WSMV), maizechlorotic dwarf virus (MCDV), barley yellow dwarf virus (BYDV), bananabunchy top virus (BBTV), and the numerous viruses listed in Table 2 ofU.S. Pat. No. 6,194,636, which is incorporated in its entirety byreference herein.

Non-limiting examples of invertebrate pests include pests capable ofinfesting the root systems of crop plants, e. g., northern corn rootworm(Diabrotica barberi), southern corn rootworm (Diabroticaundecimpunctata), Western corn rootworm (Diabrotica virgifera), cornroot aphid (Anuraphis maidiradicis), black cutworm (Agrotis ipsilon),glassy cutworm (Crymodes devastator), dingy cutworm (Feltia ducens),claybacked cutworm (Agrotis gladiaria), wireworm (Melanotus spp., Aeolusmellillus), wheat wireworm (Aeolus mancus), sand wireworm (Horistonotusuhlerii), maize billbug (Sphenophorus maidis), timothy billbug(Sphenophorus zeae), bluegrass billbug (Sphenophorus parvulus), southerncorn billbug (Sphenophorus callosus), white grubs (Phyllophaga spp.),seedcorn maggot (Delia platura), grape colaspis (Colaspis brunnea),seedcorn beetle (Stenolophus lecontei), and slender seedcorn beetle(Clivinia impressifrons), as well as the parasitic nematodes listed inTable 6 of U.S. Pat. No. 6,194,636, which is incorporated in itsentirety by reference herein.

Target genes from pests can include invertebrate genes for major spermprotein, alpha tubulin, beta tubulin, vacuolar ATPase,glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase II, chitinsynthase, cytochromes, miRNAs, miRNA precursor molecules, miRNApromoters, as well as other genes such as those disclosed in Table II ofU.S. Patent Application Publication 2004/0098761 A1, which isincorporated by reference herein. Target genes from pathogens caninclude genes for viral translation initiation factors, viralreplicases, miRNAs, miRNA precursor molecules, fungal tubulin, fungalvacuolar ATPase, fungal chitin synthase, enzymes involved in fungal cellwall biosynthesis, cutinases, melanin biosynthetic enzymes,polygalacturonases, pectinases, pectin lyases, cellulases, proteases,and other genes involved in invasion and replication of the pathogen inthe infected plant. Thus, a target gene need not be endogenous to theplant in which the recombinant DNA construct is transcribed. Arecombinant DNA construct of the invention can be transcribed in a plantand used to suppress a gene of a pathogen or pest that may infest theplant.

Specific, non-limiting examples of suitable target genes also includeamino acid catabolic genes (such as, but not limited to, the maizeLKR/SDH gene encoding lysine-ketoglutarate reductase (LKR) andsaccharopine dehydrogenase (SDH), and its homologues), maize zein genes,genes involved in fatty acid synthesis (e. g., plant microsomal fattyacid desaturases and plant acyl-ACP thioesterases, such as, but notlimited to, those disclosed in U.S. Pat. Nos. 6,426,448, 6,372,965, and6,872,872), genes involved in multi-step biosynthesis pathways, where itmay be of interest to regulate the level of one or more intermediates,such as genes encoding enzymes for polyhydroxyalkanoate biosynthesis(see, for example, U.S. Pat. No. 5,750,848); and genes encodingcell-cycle control proteins, such as proteins with cyclin-dependentkinase (CDK) inhibitor-like activity (see, for example, genes disclosedin International Patent Application Publication No. WO 05007829A2).Target genes can include genes encoding undesirable proteins (e. g.,allergens or toxins) or the enzymes for the biosynthesis of undesirablecompounds (e. g., undesirable flavor or odor components). Thus, oneembodiment of the invention is a transgenic plant or tissue of such aplant that is improved by the suppression of allergenic proteins ortoxins, e. g., a peanut, soybean, or wheat kernel with decreasedallergenicity. Target genes can include genes involved in fruitripening, such as polygalacturonase. Target genes can include geneswhere expression is preferably limited to a particular cell or tissue ordevelopmental stage, or where expression is preferably transient, thatis to say, where constitutive or general suppression, or suppressionthat spreads through many tissues, is not necessarily desired. Thus,other examples of suitable target genes include genes encoding proteinsthat, when expressed in transgenic plants, make the transgenic plantsresistant to pests or pathogens (see, for example, genes for cholesteroloxidase as disclosed in U.S. Pat. No. 5,763,245); genes where expressionis pest- or pathogen-induced; and genes which can induce or restorefertility (see, for example, the barstar/barnase genes described in U.S.Pat. No. 6,759,575); all the publications and patents cited in thisparagraph are incorporated by reference in their entirety herein.

The recombinant DNA constructs of the invention can be designed to bemore specifically suppress the target gene, by designing the genesuppression element or elements to include regions substantiallynon-identical to a non-target gene sequence. Non-target genes caninclude any gene not intended to be silenced or suppressed, either in aplant transcribing the recombinant DNA construct or in organisms thatmay come into contact with RNA transcribed from the recombinant DNAconstruct. A non-target gene sequence can include any sequence from anyspecies (including, but not limited to, non-eukaryotes such as bacteria,and viruses; fungi; plants, including monocots and dicots, such as cropplants, ornamental plants, and non-domesticated or wild plants;invertebrates such as arthropods, annelids, nematodes, and molluscs; andvertebrates such as amphibians, fish, birds, domestic or wild mammals,and even humans).

In one embodiment, the target gene is a gene endogenous to a givenspecies, such as a given plant (such as, but not limited to,agriculturally or commercially important plants, including monocots anddicots), and the non-target gene can be, e. g., a gene of a non-targetspecies, such as another plant species or a gene of a virus, fungus,bacterium, invertebrate, or vertebrate, even a human. One non-limitingexample is where the gene suppression element is designed to suppress atarget gene that is a gene endogenous to a single species (e. g.,Western corn rootworm, Diabrotica virgifera virgifera LeConte) but tonot suppress a non-target gene such as genes from related, even closelyrelated, species (e. g., Northern corn rootworm, Diabrotica barberiSmith and Lawrence, or Southern corn rootworm, Diabroticaundecimpunctata).

In other embodiments (e. g., where it is desirable to suppress a targetgene across multiple species), it may be desirable to design the genesuppression element to suppress a target gene sequence common to themultiple species in which the target gene is to be silenced. Thus, agene suppression element can be selected to be specific for one taxon(for example, specific to a genus, family, or even a larger taxon suchas a phylum, e. g., arthropoda) but not for other taxa (e. g., plants orvertebrates or mammals). In one non-limiting example of this embodiment,a gene suppression element for gene silencing can be selected so as totarget pathogenic fungi (e. g., a Fusarium spp.) but not target any genesequence from beneficial fungi.

In another non-limiting example of this embodiment, a gene suppressionelement for gene silencing in corn rootworm can be selected to bespecific to all members of the genus Diabrotica. In a further example ofthis embodiment, such a Diabrotica-targetted gene suppression elementcan be selected so as to not target any gene sequence from beneficialcoleopterans (for example, predatory coccinellid beetles, commonly knownas ladybugs or ladybirds) or other beneficial insect species.

The required degree of specificity of a gene suppression element forsuppression of a target gene depends on various factors. For example,where the gene suppression element contains DNA that transcribes to RNAfor suppressing a target gene by forming double-stranded RNA (dsRNA),factors can include the size of the smaller dsRNA fragments that areexpected to be produced by the action of Dicer, and the relativeimportance of decreasing the dsRNA's potential to suppress non-targetgenes. For example, where the dsRNA fragments are expected to be 21 basepairs in size, one particularly preferred embodiment can be to includein the gene suppression element DNA that transcribes to dsRNA and thatencodes regions substantially non-identical to a non-target genesequence, such as regions within which every contiguous fragmentincluding at least 21 nucleotides matches fewer than 21 (e. g., fewerthan 21, or fewer than 20, or fewer than 19, or fewer than 18, or fewerthan 17) out of 21 contiguous nucleotides of a non-target gene sequence.In another embodiment, regions substantially non-identical to anon-target gene sequence include regions within which every contiguousfragment including at least 19 nucleotides matches fewer than 19 (e. g.,fewer than 19, or fewer than 18, or fewer than 17, or fewer than 16) outof 19 contiguous nucleotides of a non-target gene sequence.

In some embodiments, it may be desirable to design the gene suppressionelement to include regions predicted to not generate undesirablepolypeptides, for example, by screening the gene suppression element forsequences that may encode known undesirable polypeptides or closehomologues of these. Undesirable polypeptides include, but are notlimited to, polypeptides homologous to known allergenic polypeptides andpolypeptides homologous to known polypeptide toxins. Publicly availablesequences encoding such undesirable potentially allergenic peptides areavailable, for example, the Food Allergy Research and Resource Program(FARRP) allergen database (available at allergenonline.com) or theBiotechnology Information for Food Safety Databases (available atwww.iit.edu/˜sgendel/fa.htm) (see also, for example, Gendel (1998) Adv.Food Nutr. Res., 42:63-92, which is incorporated by reference herein).Undesirable sequences can also include, for example, those polypeptidesequences annotated as known toxins or as potential or known allergensand contained in publicly available databases such as GenBank, EMBL,SwissProt, and others, which are searchable by the Entrez system(www.ncbi.nih.gov/Entrez). Non-limiting examples of undesirable,potentially allergenic peptide sequences include glycinin from soybean,oleosin and agglutinin from peanut, glutenins from wheat, casein,lactalbumin, and lactoglobulin from bovine milk, and tropomyosin fromvarious shellfish (allergenonline.com). Non-limiting examples ofundesirable, potentially toxic peptides include tetanus toxin tetA fromClostridium tetani, diarrheal toxins from Staphylococcus aureus, andvenoms such as conotoxins from Conus spp. and neurotoxins fromarthropods and reptiles (www.ncbi.nih.gov/Entrez).

In one non-limiting example, a gene suppression element is screened toeliminate those transcribable sequences encoding polypeptides withperfect homology to a known allergen or toxin over 8 contiguous aminoacids, or with at least 35% identity over at least 80 amino acids; suchscreens can be performed on any and all possible reading frames in bothdirections, on potential open reading frames that begin with ATG, or onall possible reading frames, regardless of whether they start with anATG or not. When a “hit” or match is made, that is, when a sequence thatencodes a potential polypeptide with perfect homology to a knownallergen or toxin over 8 contiguous amino acids (or at least about 35%identity over at least about 80 amino acids), is identified, the DNAsequences corresponding to the hit can be avoided, eliminated, ormodified when selecting sequences to be used in a gene suppressionelement.

Avoiding, elimination of, or modification of, an undesired sequence canbe achieved by any of a number of methods known to those skilled in theart. In some cases, the result can be novel sequences that are believedto not exist naturally. For example, avoiding certain sequences can beaccomplished by joining together “clean” sequences into novel chimericsequences to be used in a gene suppression element.

Where the gene suppression element contains DNA that transcribes to RNAfor suppressing a target gene by forming double-stranded RNA (dsRNA),applicants recognize that in some dsRNA-mediated gene silencing, it ispossible for imperfectly matching dsRNA sequences to be effective atgene silencing. For example, it has been shown that mismatches near thecenter of a miRNA complementary site has stronger effects on the miRNA'sgene silencing than do more distally located mismatches. See, forexample, FIG. 4 in Mallory et al. (2004) EMBO J., 23:3356-3364, which isincorporated by reference herein. In another example, it has beenreported that, both the position of a mismatched base pair and theidentity of the nucleotides forming the mismatch influence the abilityof a given siRNA to silence a target gene, and that adenine-cytosinemismatches, in addition to the G:U wobble base pair, were well tolerated(see Du et al. (2005) Nucleic Acids Res., 33:1671-1677, which isincorporated by reference herein). Thus, the DNA that transcribes to RNAfor suppressing a target gene by forming double-stranded RNA need notalways have 100% sequence identity with the intended target gene, butgenerally would preferably have substantial sequence identity with theintended target gene, such as about 95%, about 90%, about 85%, or about80% sequence identity with the intended target gene. One skilled in theart would be capable of judging the importance given to screening forregions predicted to be more highly specific to the first target gene orpredicted to not generate undesirable polypeptides, relative to theimportance given to other criteria, such as, but not limited to, thepercent sequence identity with the intended first target gene or thepredicted gene silencing efficiency of a given sequence. For example, itmay be desirable for a given DNA sequence for dsRNA-mediated genesilencing to be active across several species, and therefore one skilledin the art can determine that it is more important to include in thegene suppression element regions specific to the several species ofinterest, but less important to screen for regions predicted to havehigher gene silencing efficiency or for regions predicted to generateundesirable polypeptides.

Gene Expression Element: The recombinant DNA constructs of the inventioncan further include a gene expression element. Any gene or genes ofinterest can be expressed by the gene expression element, includingcoding or non-coding sequence or both, and can include naturallyoccurring sequences or artificial or chimeric sequences or both. Wherethe gene expression element encodes a protein, such constructspreferably include a functional terminator element to permittranscription and translation of the gene expression element.

In some embodiments, the recombinant DNA construct further includes agene expression element for expressing at least one gene of interest,wherein the gene expression element is located adjacent to the intron.In other embodiments, the recombinant DNA construct further includes agene expression element for expressing at least one gene of interest,wherein the gene expression element is located adjacent to the firstgene suppression element and within the intron; in such cases, the geneexpression element can be operably linked to a functional terminatorelement that is itself also within the intron. The gene of interest tobe expressed by the gene expression element can include at least onegene selected from the group consisting of a eukaryotic target gene, anon-eukaryotic target gene, and a microRNA precursor DNA sequence. Thegene of interest can include a single gene or multiple genes (such asmultiple copies of a single gene, multiple alleles of a single gene, ormultiple genes including genes from multiple species). In oneembodiment, the gene expression element can include self-hydrolyzingpeptide sequences, e. g., located between multiple sequences coding forone or more polypeptides (see, for example, the 2A and “2A-like”self-cleaving sequences from various species, including viruses,trypanosomes, and bacteria, disclosed by Donnelly et al. (2001), J. Gen.Virol., 82:1027-1041, which is incorporated herein by reference). Inanother embodiment, the gene expression element can include ribosomal“skip” sequences, e. g., located between multiple sequences coding forone or more polypeptides (see, for example, the aphthovirusfoot-and-mouth disease virus (FMDV) 2A ribosomal “skip” sequencesdisclosed by Donnelly et al. (2001), J. Gen. Virol., 82:1013-1025, whichis incorporated herein by reference).

A gene of interest can include any coding or non-coding sequence fromany species (including, but not limited to, non-eukaryotes such asbacteria, and viruses; fungi; plants, including monocots and dicots,such as crop plants, ornamental plants, and non-domesticated or wildplants; invertebrates such as arthropods, annelids, nematodes, andmolluscs; and vertebrates such as amphibians, fish, birds, and mammals.Non-limiting examples of a non-coding sequence to be expressed by a geneexpression element include, but not limited to, 5′ untranslated regions,promoters, enhancers, or other non-coding transcriptional regions, 3′untranslated regions, terminators, intron, microRNAs, microRNA precursorDNA sequences, small interfering RNAs, RNA components of ribosomes orribozymes, small nucleolar RNAs, and other non-coding RNAs. Non-limitingexamples of a gene of interest further include, but are not limited to,translatable (coding) sequence, such as genes encoding transcriptionfactors and genes encoding enzymes involved in the biosynthesis orcatabolism of molecules of interest (such as amino acids, fatty acidsand other lipids, sugars and other carbohydrates, biological polymers,and secondary metabolites including alkaloids, terpenoids, polyketides,non-ribosomal peptides, and secondary metabolites of mixed biosyntheticorigin). A gene of interest can be a gene native to the plant in whichthe recombinant DNA construct of the invention is to be transcribed, orcan be a non-native gene. A gene of interest can be a marker gene, forexample, a selectable marker gene encoding antibiotic, antifungal, orherbicide resistance, or a marker gene encoding an easily detectabletrait (e. g., phytoene synthase or other genes imparting a particularpigment to the plant), or a gene encoding a detectable molecule, such asa fluorescent protein, luciferase, or a unique polypeptide or nucleicacid “tag” detectable by protein or nucleic acid detection methods,respectively). Selectable markers are genes of interest of particularutility in identifying successful processing of constructs of theinvention.

In some embodiments of the invention, the recombinant DNA constructs aredesigned to suppress at least one endogenous gene and to simultaneouslyexpress at least one exogenous gene. In one non-limiting example, therecombinant DNA construct includes a gene suppression element forsuppressing a endogenous (maize) lysine ketoglutaratereductase/saccharopine dehydrogenase (LKR/SDH) gene and a geneexpression element for expressing an exogenous (bacterial)dihydrodipicolinic acid synthase protein, where the construct istranscribed in a maize (Zea mays) plant; such a construct would beespecially useful for providing maize with enhanced levels of lysine. Inanother non-limiting example, the recombinant DNA construct includes agene suppression element for suppressing at least one endogenous (maize)zein gene and a gene expression element for expressing an exogenous ormodified zein protein, where the construct is transcribed in a maize(Zea mays) plant; such a construct would be especially useful forproviding maize with modified zein content, e. g., zeins with modifiedamino acid composition.

Second Gene Suppression Element: In some embodiments, the recombinantDNA construct further includes a second gene suppression element forsuppressing at least one second target gene, wherein the second genesuppression element is located adjacent to the intron. The second genesuppression element can include any element as described above under“Gene Suppression Elements”. In these embodiments, where the constructincludes a functional terminator element, the construct can be designedso that the first gene suppression element, which is embedded in theintron, preferably causes nuclear suppression of the first target gene,whereas the second gene suppression element preferably causesextra-nuclear or cytoplasmic suppression of the second target gene. Thesecond target gene can be any gene or genes as described above under theheading “Target Genes”, and can include coding or non-coding sequence orboth. The second target gene or genes can be endogenous or exogenous tothe plant in which the recombinant DNA construct is transcribed, and caninclude multiple target genes.

Methods of Gene Suppression and Methods for Screening for Traits: Thepresent invention provides a method of effecting gene suppression,including (a) providing a transgenic plant comprising a regeneratedplant prepared from a transgenic plant cell containing a recombinant DNAconstruct for plant transformation including a promoter operably linkedto a first gene suppression element for suppressing at least one firsttarget gene, wherein said first gene suppression element is embedded inan intron flanked on one or on both sides by non-protein-coding DNA, ora progeny plant of said regenerated plant; and (b) transcribing saidrecombinant DNA construct in said transgenic plant; wherein saidtranscribing produces RNA that is capable of suppressing said at leastone first target gene in said transgenic plant, whereby said at leastone first target gene is suppressed relative to its expression in theabsence of transcription of said recombinant DNA construct.

In some embodiments, the at least one first target gene is at least onegene selected from the group consisting of a gene native to saidtransgenic plant, a transgene in said transgenic plant, a gene native toa viral, a bacterial, a fungal, or an invertebrate pest or pathogen ofsaid transgenic seed or of said transgenic plant, a microRNA precursorsequence, and a microRNA promoter. The at least one first target genecan be multiple target genes. In other embodiments, the gene suppressionis nuclear suppression of a microRNA precursor sequence or a microRNApromoter. Gene suppression by the method of the invention can bespatially specific, temporally specific, developmentally specific, orinducible gene suppression. In another embodiment of the method, therecombinant DNA construct further includes a gene expression element forexpressing at least one gene of interest, wherein the gene expressionelement is located outside of (e. g., adjacent to) the intron, andwherein the gene suppression is effected with concurrent expression ofthe at least one gene of interest in the transgenic plant.

In one preferred embodiment of the method, the resulting genesuppression is non-systemic suppression of a gene native to thetransgenic plant or a transgene in the transgenic plant, for example, toprovide non-systemic, tissue-specific suppression of at least one targetgene in the transgenic plant, which can be useful, for example, forlimiting gene suppression to specific tissue, such as in seeds or rootsin plants, wherein the target gene can be native to the transgenic plantin which the construct is transcribed or native to a pest or pathogen ofsaid plant. In such embodiments, it is preferred that the transcribableheterologous DNA is transcribed to RNA that remains in the nucleus, forexample, to a messenger RNA (mRNA) that lacks processing signals such aspplyadenylation for transport of the mRNA to the cytoplasm. In oneparticular example of this embodiment, the gene suppression isnon-systemic, nuclear suppression of a microRNA precursor DNA sequenceor of a microRNA promoter. The method can employ the recombinant DNAconstructs of this invention to modify the lipid, protein, carbohydrate,or amino acid composition or content of plant seeds by non-systemicallysuppressing enzymes in biosynthetic pathways for such components. In anon-limiting specific example, transgenic maize having recombinant DNAfor suppressing lysine ketoglutarate reductase (LKR/SDH) can be producedusing a recombinant DNA construct of this invention consisting of anendosperm-specific or a seed-specific promoter operably linked to, anintron containing, for example, tandem copies of anti-sense oriented DNAfrom the maize endogenous gene encoding LKR/SDH. Suppression of LKR/SDHis non-systemic (depending on the promoter, limited to the endosperm orto the seed), and seed from such a transgenic maize plant with therecombinant DNA construct will have increased lysine as compared to seedof substantially equivalent genotype but without the recombinant DNA.

The present invention further provides a method of concurrentlyeffecting gene suppression of at least one target gene and geneexpression of at least one gene of interest, including growing atransgenic plant from a transgenic seed having in its genome arecombinant DNA construct for suppressing at least one first targetgene, including DNA capable of initiating transcription in a plant andoperably linked to a first transcribable heterologous DNA, wherein thefirst transcribable heterologous DNA is embedded in an intron, andwherein the recombinant DNA construct further includes a gene expressionelement for expressing the at least one gene of interest, the geneexpression element being located adjacent to the intron, and wherein,when the recombinant DNA construct is transcribed in the transgenicplant, transcribed RNA that is capable of suppressing the at least onefirst target gene and transcribed RNA encoding the at least one gene ofinterest are produced, whereby the at least one first target gene issuppressed relative to its expression in the absence of transcription ofthe recombinant DNA construct and the at least one gene of interest isexpressed. The transcribed RNA that is capable of suppressing the atleast one first target gene is transcribed from the intron-embeddedfirst transcribable heterologous DNA. The transcribed RNA encoding theat least one gene of interest is transcribed from the gene expressionelement. Where the transcribed RNA encoding the at least one gene ofinterest includes coding region for a protein to be expressed, it ispreferably transcribed as RNA capable of transport into the cytoplasmfor translation. The intron-embedded first transcribable heterologousDNA can be designed to suppress a single or multiple target genes. Thegene expression element can be designed to express a single or multipletarget genes. Optionally, the recombinant DNA construct can include asecond transcribable heterologous DNA for suppression at least onesecond target gene, wherein the second transcribable heterologous DNA istranscribed into RNA capable of transport into the cytoplasm; in suchembodiments of the method, the at least one first target gene ispreferably suppressed by nuclear suppression, and the at least onesecond target gene is preferably suppressed by cytoplasmic suppression.

In one embodiment of the method, transgenic plants are produced thathave a modified nutritional content, or that produce seed having amodified nutritional content. In particularly preferred embodiment, themethod is useful for providing transgenic maize producing seed withenhanced levels of lysine, tryptophan, methionine, oil, or a combinationof any of these. In one non-limiting example, the method makes use of arecombinant DNA construct that includes (a) a gene suppression element(embedded in an intron flanked on one or both sides bynon-protein-coding DNA) for suppressing a endogenous (maize) lysineketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) gene, and,optionally, (b) a gene expression element for expressing an exogenous(e. g., a bacterial) dihydrodipicolinic acid synthase protein, where theconstruct is transcribed in a maize (Zea mays) plant. This methodpreferably provides transgenic maize producing seed with enhanced levelsof lysine (free or protein-bound or both). In another non-limitingexample of the method, the recombinant DNA construct includes a genesuppression element (embedded in an intron flanked on one or both sidesby non-protein-coding DNA) for suppressing at least one endogenous(maize) zein synthesis gene (e. g., an alpha-zein, such as a19-kiloDalton alpha-zein or a 22-kiloDalton alpha-zein, or a geneencoding any one or more of the alpha-, beta-, gamma-, and delta-zeins)and optionally for suppressing an endogenous (maize) lysine catabolicenzyme gene (lysine ketoglutarate reductase/saccharopine dehydrogenaseor LKR/SDH), and a gene expression element for expressing an exogenouslysine synthesis gene sequence encoding enzymes for synthesis of lysineor its precursors (e. g., aspartate kinase (AK) and dihydrodipicolinicacid synthase (DHDPS), and homologues of these genes). This methodpreferably provides transgenic maize producing seed with enhanced levelsof lysine (free or protein-bound or both), and more preferably providestransgenic maize producing seed with enhanced levels of two or more oflysine, tryptophan, and oil. Also preferred are methods using similarrecombinant DNA constructs to transform maize, where, for example, thegene expression element is used to express other biosynthetic genes ofinterest, such as asparagine synthase or a modified zein or otherstorage protein, wherein the resulting transgenic maize produces seedcontaining modified free amino acid or protein content, preferably withenhanced levels of lysine, tryptophan, methionine, oil, or a combinationof these.

The present invention further provides a method of concurrentlyeffecting gene suppression of at least one target gene and geneexpression of at least one gene of interest, including: (a) providing atransgenic plant comprising a regenerated plant prepared from atransgenic plant cell containing a recombinant DNA construct for planttransformation including a promoter operably linked to a first genesuppression element for suppressing at least one first target gene,wherein the first gene suppression element is embedded in an intronflanked on one or on both sides by non-protein-coding DNA, or a progenyplant of the regenerated plant, wherein the recombinant DNA constructfurther includes a gene expression element for expressing the at leastone gene of interest and the gene expression element is located adjacentto the intron; and (b) transcribing the recombinant DNA construct in thetransgenic plant, wherein, when the recombinant DNA construct istranscribed in the transgenic plant, transcribed RNA that is capable ofsuppressing the at least one first target gene and transcribed RNAencoding the at least one gene of interest are produced, whereby the atleast one first target gene is suppressed relative to its expression inthe absence of transcription of the recombinant DNA construct and the atleast one gene of interest is concurrently expressed.

The present invention also provides a method of screening for traits ina transgenic plant resulting from suppression of an endogenous gene,wherein the method includes: (a) providing a transgenic plant includes aregenerated plant prepared from a transgenic plant cell containing arecombinant DNA construct for plant transformation including a promoteroperably linked to a first gene suppression element for suppressing atleast one first target gene, wherein the first gene suppression elementis embedded in an intron flanked on one or on both sides bynon-protein-coding DNA, or a progeny plant of the regenerated plant; (b)transcribing the recombinant DNA construct in said transgenic plant; and(c) analyzing the transgenic plant for the traits. The method canoptionally further include screening for transcription of the genesuppression element. In some embodiments of the method wherein therecombinant DNA construct further includes at least one gene expressionelement, the screening can optionally further include detection ofexpression of a gene encoded by the gene expression element.

The methods of the invention make use of procedures to introduce therecombinant DNA constructs into a transgenic plant cell, and theproduction of transgenic plants or progeny plants from such cells. Suchprocedures are described under the heading “Making and Using TransgenicPlant Cells and Plants”. Detecting or measuring the gene suppression (orconcurrent gene expression) obtained by transcription of the constructcan be achieved by any suitable methods, including protein detectionmethods (e. g., western blots, ELISAs, and other immunochemicalmethods), measurements of enzymatic activity, or nucleic acid detectionmethods (e. g., Southern blots, northern blots, PCR, RT-PCR, fluorescentin situ hybridization,). Such methods are well known to those ofordinary skill in the art as evidenced by the numerous handbooksavailable; see, for example, Joseph Sambrook and David W. Russell,“Molecular Cloning: A Laboratory Manual” (third edition), Cold SpringHarbor Laboratory Press, NY, 2001; Frederick M. Ausubel et al. (editors)“Short Protocols in Molecular Biology” (fifth edition), John Wiley andSons, 2002; John M. Walker (editor) “Protein Protocols Handbook” (secondedition), Humana Press, 2002; and Leandro Peña (editor) “TransgenicPlants: Methods and Protocols”, Humana Press, 2004, which areincorporated by reference herein.

Other suitable methods for detecting or measuring gene suppression (orconcurrent gene expression) include measurement of any other trait thatis a direct or proxy indication of gene suppression (or concurrent geneexpression) in the plant in which the construct is transcribed, relativeto one in which the construct is not transcribed, e. g., gross ormicroscopic morphological traits, growth rates, yield, reproductive orrecruitment rates, resistance to pests or pathogens, or resistance tobiotic or abiotic stress (e. g., water deficit stress, salt stress,nutrient stress, heat or cold stress). Such methods can use directmeasurements of a phenotypic trait or proxy assays (e. g., plant partassays such as leaf or root assays to determine tolerance of abioticstress).

III. Recombinant DNA Constructs for Suppressing Production of MaturemiRNA and Methods of Use Thereof

Another aspect of the invention provides a recombinant DNA construct forsuppressing production of mature microRNA in a cell, including apromoter element operably linked to a gene suppression element forsuppression of at least one target sequence selected from the at leastone target microRNA precursor or a promoter of the at least one targetmicroRNA precursor or both. In one non-limiting embodiment, the targetsequence includes nucleotides of a loop region of at least one targetmicroRNA precursor (that is, at least some nucleotides in anysingle-stranded region forming a loop-like or gap-like domain in astem-loop RNA structure of a pri-miRNA or a pre-miRNA). Target microRNAprecursor DNA sequences can be native (endogenous) to the cell (e. g., acell of a plant or animal) in which the recombinant DNA construct of theinvention is transcribed, or can be native to a pest or pathogen of theplant or animal in which the recombinant DNA construct of the inventionis transcribed.

Using constructs of the invention, suppression of production of maturemiRNA can occur in the nucleus or in the cytoplasm or in both. In manypreferred embodiments, particularly (but not limited to) embodimentswhere the suppression occurs in a plant cell, suppression preferablyoccurs wholly or substantially in the nucleus, and the gene suppressionelement is preferably transcribed to RNA lacking functional nuclearexport signals. In these embodiments, the RNA transcribed from such agene suppression element preferably remains in the nucleus and resultsin enhanced nuclear suppression of production of mature miRNA; such agene suppression element is preferably characterized by at least one ofthe following: (a) at least one of a functional polyadenylation signaland a functional polyadenylation site is absent; (b) a 3′ untranslatedregion is absent; (c) a self-splicing ribozyme is located adjacent toand 3′ to the suppression element; and/or (d) the suppression element isembedded in an intron, preferably an intron flanked on one or on bothsides by non-protein-coding DNA.

The recombinant DNA construct for suppressing production of maturemicroRNA in a cell includes at least one gene suppression elementselected from the group consisting of: (a) DNA that includes at leastone anti-sense DNA segment that is anti-sense to at least one segment ofthe at least one target sequence; (b) DNA that includes multiple copiesof at least one anti-sense DNA segment that is anti-sense to at leastone segment of the at least one target sequence; (c) DNA that includesat least one sense DNA segment that is at least one segment of the atleast one target sequence; (d) DNA that includes multiple copies of atleast one sense DNA segment that is at least one segment of the at leastone target sequence; (e) DNA that transcribes to RNA for suppressing theat least one first target sequence by forming double-stranded RNA andincludes at least one anti-sense DNA segment that is anti-sense to atleast one segment of the at least one target sequence and at least onesense DNA segment that is at least one segment of the at least onetarget sequence; (f) DNA that transcribes to RNA for suppressing the atleast one first target sequence by forming a single double-stranded RNAand includes multiple serial anti-sense DNA segments that are anti-senseto at least one segment of the at least one target sequence and multipleserial sense DNA segments that are at least one segment of the at leastone target sequence; (g) DNA that transcribes to RNA for suppressing theat least one first target sequence by forming multiple double strands ofRNA and includes multiple anti-sense DNA segments that are anti-sense toat least one segment of the at least one target sequence and multiplesense DNA segments that are at least one segment of the at least onetarget sequence, and wherein the multiple anti-sense DNA segments andthe multiple sense DNA segments are arranged in a series of invertedrepeats; (h) DNA that includes nucleotides derived from a miRNA (whichcan be an animal, plant, or viral miRNA and is preferably a viral or ananimal miRNA where the construct is to be transcribed in an animal cell,and preferably a viral or a plant miRNA where the construct is to betranscribed in a plant cell); and (i) DNA that includes nucleotides of asiRNA; (j) DNA that transcribes to an RNA aptamer capable of binding toa ligand; and (k) DNA that transcribes to an RNA aptamer capable ofbinding to a ligand, and DNA that transcribes to regulatory RNA capableof regulating expression of the first target gene, wherein theregulation is dependent on the conformation of the regulatory RNA, andthe conformation of the regulatory RNA is allosterically affected by thebinding state of the RNA aptamer. In some embodiments, the genesuppression element suppresses multiple target microRNA precursors ormultiple microRNA promoters or a combination of both. In someembodiments, the target sequence includes nucleotides of a loop regionof the at least one target microRNA precursor.

There the recombinant DNA construct is to be transcribed in an animalcell, the promoter includes a promoter element functional in an animal,and the at least one target microRNA precursor is endogenous to theanimal or a eukaryotic pest or pathogen of the animal. Where therecombinant DNA construct is to be transcribed in a plant cell, thepromoter element is functional in a plant, and the at least one targetmicroRNA precursor is endogenous to the plant or to a eukaryotic pest oreukaryotic pathogen of the plant. In various embodiments, therecombinant DNA construct includes a promoter element which can beselected from the group consisting of a constitutive promoter, aspatially specific promoter, a temporally specific promoter, adevelopmentally specific promoter, and an inducible promoter.

In various embodiments, the recombinant DNA construct for suppressingproduction of mature microRNA in a cell optionally includes at least oneof: (a) at least one T-DNA border; (b) spacer DNA; (c) a gene expressionelement for expressing at least one gene of interest; and (d) a secondgene suppression element for suppressing at least one second targetgene, wherein the second gene suppression element is located adjacent tothe intron. In various embodiments, the recombinant DNA construct isfurther characterized by any of the following conditions: (a) theterminator element includes a functional polyadenylation signal andpolyadenylation site; or (b) at least one of a functionalpolyadenylation signal and a functional polyadenylation site is absentin the terminator element; or (c) a 3′ untranslated region is absent.

The invention further provides a method of effecting suppression ofmature microRNA production in a eukaryotic cell, including transcribingin a eukaryotic cell a recombinant DNA construct for suppressingproduction of mature microRNA in a cell, including a promoter elementoperably linked to a gene suppression element for suppression of atleast one target sequence selected from the at least one target microRNAprecursor or a promoter of the at least one target microRNA precursor orboth, whereby mature microRNA production is suppressed relative to itsproduction in the absence of transcription of the recombinant DNAconstruct. In one preferred embodiment of the method, the suppression isnuclear suppression, and the suppression element is transcribed in thecell to RNA lacking functional nuclear export signals. The suppressionelement suppresses at least one target sequence selected from at leastone target microRNA precursor molecule or a promoter of the at least onemicroRNA precursor molecule, or both. The method can includetranscription of the recombinant DNA construct in a cell of an animal,wherein the at least one target microRNA precursor is endogenous to theanimal or a eukaryotic or viral pest or pathogen of the animal. Themethod can include transcription of the recombinant DNA construct in acell of a plant, wherein the at least one target microRNA precursor isendogenous to the plant or a eukaryotic or viral pest or pathogen of theplant. In various embodiments, the recombinant DNA construct furtherincludes a gene expression element for expressing at least one gene ofinterest, wherein the suppression of mature microRNA production iseffected with concurrent expression of the at least one gene of interestin the cell.

In preferred embodiments, the mature miRNA to be suppressed is a plantmiRNA in a plant cell. Suppression can be of a consensus sequence ofmultiple mature miRNAs or multiple miRNA precursors, or of a miRNApromoter that promotes transcription of multiple miRNAs, or of aconsensus sequence of multiple miRNA promoters. In preferredembodiments, the mature miRNA is a miRNA of a crop plant, such as, butnot limited to, a miRNA of any of the plant species enumerated under theheading “Transgenic Plants”. Especially preferred are methods where themature miRNA to be suppressed is a maize or soybean mature microRNA. Inpreferred embodiments, the target microRNA precursor molecule is derivedfrom the fold-back structure of a crop plant mature miRNA, such as amaize or soybean MIR sequence selected from the MIR sequences identifiedin Tables 3, 4, 5, 6, 9, and 10, and their complements. In specificallyclaimed embodiments, the target microRNA precursor molecule is derivedfrom the fold-back structure of a maize or soybean MIR sequence selectedfrom the group consisting of SEQ ID NO. 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 28, 30, 32, 34, 38, 39, 43, 44, 227, 228, 236, 239, 242,245, 248, and 249, and their complements.

Promoters and other elements useful in the recombinant DNA constructsfor suppressing production of mature microRNA in a cell are described indetail under the headings “Gene Suppression Elements”, “PromoterElements”, “Introns”, “Terminator Elements”, “T-DNA Borders”, “SpacerDNA”, and “Gene Expression Elements”, and elsewhere in this disclosure.Techniques for making and using recombinant DNA constructs of theinvention, for making transgenic plant cells and transgenic plants,seeds, and progeny plants, and for assaying the effects of transcribingthe recombinant DNA constructs, are described in detail under theheadings “Making and Using Recombinant DNA Constructs”, “Making andUsing Transgenic Plant Cells and Transgenic Plants”, and elsewhere inthis disclosure.

IV. Engineered Heterologous miRNA for Controlling Gene Expression

Engineered miRNAs and trans-acting siRNAs (ta-siRNAs) are useful forgene suppression with increased specificity. The invention provides arecombinant DNA construct including a transcribable engineered miRNAprecursor designed to suppress a target sequence, wherein thetranscribable engineered miRNA precursor is derived from the fold-backstructure of a MIR gene, preferably a maize or soybean MIR sequenceselected from the group consisting of the MIR sequences identified inTables 3, 4, 5, 6, 9, and 10, and their complements. In specificallyclaimed embodiments, the transcribable engineered miRNA precursor isderived from the fold-back structure of a maize or soybean MIR sequenceselected from the group consisting of SEQ ID NO. 6, 7, 8, 9, 10, 12, 14,16, 18, 20, 22, 24, 28, 30, 32, 34, 38, 39, 43, 44, 227, 228, 236, 239,242, 245, 248, and 249, and their complements. These miRNA precursorsare also useful for directing in-phase production of siRNAs (e. g.,heterologous sequence designed to be processed in a trans-acting siRNAsuppression mechanism in planta). The invention further provides amethod to suppress expression of a target sequence in a plant cell,including transcribing in a plant cell a recombinant DNA constructincluding a transcribable engineered miRNA precursor designed tosuppress a target sequence, wherein the transcribable engineered miRNAprecursor is derived from the fold-back structure of a MIR gene,preferably a maize or soybean MIR sequence selected from the groupconsisting of the MIR sequences identified in Tables 2, 3, and 4, andtheir complements, whereby expression of the target sequence issuppressed relative to its expression in the absence of transcription ofthe recombinant DNA construct. In specifically claimed embodiments, thetranscribable engineered miRNA precursor is derived from the fold-backstructure of a maize or soybean MIR sequence selected from the groupconsisting of SEQ ID NO. 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 28,30, 32, 34, 38, 39, 43, 44, 227, 228, 236, 239, 242, 245, 248, and 249,and their complements.

The mature miRNAs produced, or predicted to be produced, from thesemiRNA precursors may be engineered for use in suppression of a targetgene, e. g., in transcriptional suppression by the miRNA, or to directin-phase production of siRNAs in a trans-acting siRNA suppressionmechanism (see Allen et al. (2005) Cell, 121:207-221, Vaucheret (2005)Science STKE, 2005:pe43, and Yoshikawa et al. (2005) Genes Dev.,19:2164-2175, all of which are incorporated by reference herein). PlantmiRNAs generally have near-perfect complementarity to their targetsequences (see, for example, Llave et al. (2002) Science, 297:2053-2056,Rhoades et al. (2002) Cell, 110:513-520, Jones-Rhoades and Bartel (2004)Mol. Cell, 14:787-799, all of which are incorporated by referenceherein). Thus, the mature miRNAs can be engineered to serve as sequencesuseful for gene suppression of a target sequence, by replacingnucleotides of the mature miRNA sequence with nucleotides of thesequence that is targetted for suppression; see, for example, methodsdisclosed by Parizotto et al. (2004) Genes Dev., 18:2237-2242 andespecially U.S. Patent Application Publications 2004/0053411A1,2004/0268441A1, 2005/0144669, and 2005/0037988 all of which areincorporated by reference herein. When engineering a novel miRNA totarget a specific sequence, one strategy is to select within the targetsequence a region with sequence that is as similar as possible to thenative miRNA sequence. Alternatively, the native miRNA sequence can bereplaced with a region of the target sequence, preferably a region thatmeets structural and thermodynamic criteria believed to be important formiRNA function (see, for example, U.S. Patent Application Publication2005/0037988). Sequences are preferably engineered such that the numberand placement of mismatches in the stem structure of the fold-backregion or pre-miRNA is preserved. Thus, an engineered miRNA orengineered miRNA precursor can be derived from any of the mature miRNAsequences, or their corresponding miRNA precursors (including thefold-back portions of the corresponding MIR genes) disclosed herein. Theengineered miRNA precursor can be cloned and expressed (transiently orstably) in a plant cell or tissue or intact plant.

Promoters and other elements useful in the recombinant DNA constructsincluding a transcribable engineered miRNA precursor designed tosuppress a target sequence are described in detail under the headings“Gene Suppression Elements”, “Promoter Elements”, “Introns”, “TerminatorElements”, “T-DNA Borders”, “Spacer DNA”, and “Gene ExpressionElements”, and elsewhere in this disclosure. Techniques for making andusing recombinant DNA constructs of the invention, for making transgenicplant cells containing the recombinant DNA constructs and transgenicplants, seeds, and progeny plants derived therefrom, and for assayingthe effects of transcribing the recombinant DNA constructs, aredescribed in detail under the headings “Making and Using Recombinant DNAConstructs”, “Making and Using Transgenic Plant Cells and TransgenicPlants”, and elsewhere in this disclosure.

V. Recombinant DNA Constructs Including Exogenous miRNA RecognitionSites and Methods for Use Thereof

One aspect of the invention provides a recombinant DNA constructincluding transcribable DNA that transcribes to RNA including (a) atleast one exogenous miRNA recognition site recognizable by a maturemiRNA expressed in a specific cell of a multicellular eukaryote, and (b)target RNA to be suppressed in the specific cell, whereby said targetRNA is expressed in cells other than said specific cell. Themulticellular eukaryote can be any multicellular eukaryote (e. g.,plant, animal, or fungus), and is preferably a plant or an animal. Theconstructs are prepared by methods known in the art, for example, asdisclosed below under the heading “Making and Using Recombinant DNAConstructs of the Invention”.

Generally, the recombinant DNA construct includes a promoter operablylinked to the transcribable DNA. Suitable promoters include any promoterthat is capable of transcribing DNA in the cell where transcription isdesired, and are generally promoters functional in a eukaryotic cell, e.g., the promoters listed below under the heading “Promoter Elements”.Where the specific cell is an animal cell, the promoter is a promoterfunctional in the animal cell. Where the specific cell is a plant cell,the promoter is a promoter functional in the plant cell. In oneembodiment of the invention, the promoter is preferably a constitutivepromoter or a promoter that allows expression in cells not limited tothe specific cell in which expression of the target RNA is to besuppressed. The recombinant DNA construct can optionally include aterminator, e. g., a functional terminator that allows polyadenylationof the transcript.

Mature miRNA: By mature miRNA is meant the small RNA processed from amiRNA precursor (e. g., pri-miRNA or pre-miRNA), that is capable ofrecognizing and binding to a specific sequence (“miRNA recognitionsite”) within an RNA transcript, and guiding the cleavage of thattranscript. In one preferred, non-limiting embodiment of the invention,the mature miRNA is a crop plant miRNA, such as a maize miRNA or a soymiRNA. Non-limiting examples of specific miRNAs are provided in theExamples.

Target RNA: The target RNA is any RNA of interest, and can include atleast one of non-coding RNA, a suppression element; and a geneexpression element, or any combination of these. Non-coding RNA caninclude RNA that functions as a suppression element (such as thosedescribed under the heading “Gene Suppression Elements”) as well as RNAwith a secondary structure conferring upon it a desired function, e. g.,RNA ribozymes or RNA aptamers that can bind a specific ligand. Thetarget RNA can include a gene expression element (described under theheading “Gene Expression Elements”) and can include coding or non-codingsequence from any species.

miRNA Recognition Site: The at least one miRNA recognition site isexogenous, that is, occurring in other than a naturally occurring ornative context. One or more (identical or different) exogenous miRNArecognition sites can be variously located in the recombinant DNAconstruct: (a) in the 5′ untranslated region of the target RNA, or (b)in the 3′ untranslated region of the target RNA, or (c) within thetarget RNA. Inclusion of the exogenous miRNA recognition site within acoding region may be constrained by the requirements of the amino acidsequence, but is possible if the inclusion does not produce translatedpolypeptides with undesirable characteristics (e. g., loss or decreaseof function). Any miRNA recognition site may be used in carrying out theinvention; particularly preferred are any of the miRNA recognition sitesprovided in Tables 8, 11, and 12, and specifically claimed are the miRNArecognition sites having SEQ ID NOS. 64-219 and 250-346.

In one non-limiting embodiment, it may be desirable to express thetarget RNA under a non-specific (e. g., a “strong” constitutivepromoter) throughout most cells, but not in specific cells, of amulticellular eukaryote such as a plant. Thus, the at least oneexogenous miRNA recognition site is generally chosen according toknowledge of spatial or temporal expression of the corresponding maturemiRNA that recognizes and binds to the miRNA recognition site.

Cleavage of a target RNA transcript and the subsequent suppression ofthe target RNA is dependent on base pairing between the mature miRNA andits cognate miRNA recognition site. Thus, the at least one exogenousmiRNA recognition site is designed to have sufficient sequencecomplementarity to the mature miRNA to allow recognition and binding bythe mature miRNA. In plants, sequence complementarity of a miRNA and itsrecognition site is typically high, e. g., perfect complementaritybetween 19, 20, or 21 out of 21 nucleotides (in the case of a maturemiRNA that is 21 nucleotides in length), that is, complementarity ofabout 90% or greater. A similar degree of complementarity is preferablefor recognition sites for plant miRNAs of any length (e. g., 20, 21, 22,23, and 24 nucleotides). The sequence requirements for mature miRNAbinding to a recognition site, and methods for predicting miRNA bindingto a given sequence, are discussed, for example, in Llave et al. (2002)Science, 297:2053-2056, Rhoades et al. (2002) Cell, 110:513-520,Jones-Rhoades and Bartel (2004) Mol. Cell, 14:787-799, Schwab et al.(2005) Developmental Cell, 8:517-527, and Xie et al. (2005) PlantPhysiol., 138:2145-2154, all of which are incorporated by referenceherein. When designing a miRNA recognition site as well as its exactlocation in or adjacent to a target RNA, it is also preferable to avoidsequences that have undesirable characteristics, such sequences encodingundesirable polypeptides, as described under the heading “Target Genes”.When designing target RNA as a transgene to be expressed, theunintentional introduction of an exogenous miRNA recognition site ispreferably avoided where suppression by a mature miRNA is not desired.

One preferred aspect of the invention includes a transgenic plant cellor a transgenic plant containing in its genome the recombinant DNAconstruct including at least one exogenous miRNA recognition site andtarget RNA. Suitable transgenic plants include a regenerated plantprepared from a transgenic plant cell having in its genome therecombinant DNA construct including at least one exogenous miRNArecognition site and target RNA, or a progeny plant of such aregenerated plant; progeny plants include plants of any developmentalstage (including seeds) and include hybrid progeny plants. One preferredembodiment is a transgenic crop plant wherein the mature miRNA thatrecognizes the exogenous miRNA recognition site is a maize or soybeanmiRNA (e. g., a miRNA derived from the fold-back structure of a maize orsoybean MIR sequence selected from the MIR sequences identified inTables 3, 4, 5, 6, 9, and 10, and their complements, or morespecifically, a MIR sequence selected from SEQ ID NO. 6, 7, 8, 9, 10,12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 38, 39, 43, 44, 227, 228,236, 239, 242, 245, 248, and 249, and their complements).

These constructs are useful in methods, as disclosed and claimed herein,for suppressing expression of a target RNA in a specific cell of amulticellular eukaryote, including transcribing in the multicellulareukaryote a recombinant DNA construct including a promoter operablylinked to DNA that transcribes to RNA including: (a) at least oneexogenous miRNA recognition site recognizable by a mature miRNAexpressed in a specific cell, and (b) target RNA to be suppressed in thespecific cell, wherein the mature miRNA guides cleavage of target RNA inthe specific cell, whereby expression of the target RNA is suppressed inthe specific cell relative to its expression in cells lacking expressionof the mature miRNA. Suitable multicellular eukaryotes include plants(e. g., mosses, ferns, monocots, and dicots) and animals (includingmammals and other vertebrates). Where the multicellular eukaryote is aplant, the mature miRNA is preferably a plant mature miRNA; in someembodiments, the mature miRNA is preferably a mature miRNA from a cropplant such as, but not limited to, maize or soy (e. g., a miRNA derivedfrom the fold-back structure of a maize or soybean MIR sequence selectedfrom the MIR sequences identified in Tables 3, 4, 5, 6, 9, and 10, andtheir complements, or more specifically, a MIR sequence selected fromSEQ ID NO. 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34,38, 39, 43, 44, 227, 228, 236, 239, 242, 245, 248, and 249, and theircomplements).

In some embodiments, the recombinant DNA construct further includes agene expression element for expressing at least one gene of interest (asdescribed in detail below under “Gene Expression Element”), wherein theexpression of the target RNA is suppressed with concurrent expression ofthe at least one gene of interest in the specific cell. In otherembodiments, the target RNA includes a gene suppression element embeddedin an intron, preferably an intron flanked on one or on both sides bynon-protein-coding DNA, as described under “II. Recombinant DNAConstructs Containing Introns and Gene Suppression Elements”.

Promoters and other elements useful in the recombinant DNA constructsincluding at least one exogenous miRNA recognition site and target RNAare described in detail under the headings “Gene Suppression Elements”,“Promoter Elements”, “Introns”, “Terminator Elements”, “T-DNA Borders”,“Spacer DNA”, and “Gene Expression Elements”, and elsewhere in thisdisclosure. Techniques for making and using recombinant DNA constructsof the invention, for making transgenic plant cells containing therecombinant DNA constructs and transgenic plants, seeds, and progenyplants derived therefrom, and for assaying the effects of transcribingthe recombinant DNA constructs, are described in detail under theheadings “Making and Using Recombinant DNA Constructs”, “Making andUsing Transgenic Plant Cells and Transgenic Plants”, and elsewhere inthis disclosure.

Making and Using Recombinant DNA Constructs: The recombinant DNAconstructs of the present invention can be made by any method suitableto the intended application, taking into account, for example, the typeof expression desired and convenience of use in the plant in which theconstruct is to be transcribed. General methods for making and using DNAconstructs and vectors are well known in the art and described in detailin, for example, handbooks and laboratory manuals including Sambrook andRussell, “Molecular Cloning: A Laboratory Manual” (third edition), ColdSpring Harbor Laboratory Press, NY, 2001, which is incorporated hereinby reference. An example of useful technology for building DNAconstructs and vectors for transformation is disclosed in U.S. PatentApplication Publication 2004/0115642 A1, incorporated herein byreference. DNA constructs can also be built using the GATEWAY™ cloningtechnology (available from Invitrogen Life Technologies, Carlsbad,Calif.), which uses the site-specific recombinase LR cloning reaction ofthe Integrase/att system from bacteriophage lambda vector construction,instead of restriction endonucleases and ligases. The LR cloningreaction is disclosed in U.S. Pat. Nos. 5,888,732 and 6,277,608, and inU.S. Patent Application Publications 2001/283529, 2001/282319 and2002/0007051, all of which are incorporated herein by reference. TheGATEWAY™ Cloning Technology Instruction Manual, which is also suppliedby Invitrogen, provides concise directions for routine cloning of anydesired DNA into a vector comprising operable plant expression elements.Another alternative vector fabrication method employsligation-independent cloning as disclosed by Aslandis et al. (1990)Nucleic Acids Res., 18:6069-6074 and Rashtchian et al. (1992) Biochem.,206:91-97, where a DNA fragment with single-stranded 5′ and 3′ ends isligated into a desired vector which can then be amplified in vivo.

In certain embodiments, the DNA sequence of the recombinant DNAconstruct includes sequence that has been codon-optimized for the plantin which the recombinant DNA construct is to be expressed. For example,a recombinant DNA construct to be expressed in a plant can have all orparts of its sequence (e. g., the first gene suppression element or thegene expression element) codon-optimized for expression in a plant. See,e. g., U.S. Pat. No. 5,500,365; De Amicis and Marchetti (2000) NucleicAcid Res., 28:3339-3346, which are incorporated by reference herein.

In certain embodiments, the DNA sequence of the recombinant DNAconstruct includes sequence that has been codon-optimized for the cell(e. g., an animal, plant, or fungal cell) in which the construct is tobe expressed. For example, a construct to be expressed in a plant cellcan have all or parts of its sequence (e. g., the first gene suppressionelement or the gene expression element) codon-optimized for expressionin a plant. See, for example, U.S. Pat. No. 5,500,365; De Amicis andMarchetti (2000) Nucleic Acid Res., 28:3339-3346, which are incorporatedby reference herein.

Making and Using Transgenic Plant Cells and Transgenic Plants: Theinvention provides and claims a transgenic plant cell having in itsgenome any of the recombinant DNA constructs presently disclosed. Thetransgenic plant cell can be an isolated plant cell (e. g., individualplant cells or cells grown in or on an artificial culture medium), orcan be a plant cell in undifferentiated tissue (e. g., callus or anyaggregation of plant cells). The transgenic plant cell can be a plantcell in at least one differentiated tissue selected from the groupconsisting of leaf (e. g., petiole and blade), root, stem (e. g., tuber,rhizome, stolon, bulb, and corn) stalk (e. g., xylem, phloem), wood,seed, fruit (e. g., nut, grain, fleshy fruits), and flower (e. g.,stamen, filament, anther, pollen, carpel, pistil, ovary, ovules). Theinvention further provides a transgenic plant having in its genome anyof the recombinant DNA constructs presently disclosed, including aregenerated plant prepared from the transgenic plant cells claimedherein, or a progeny plant (which can be a hybrid progeny plant) of theregenerated plant, or seed of such a transgenic plant. Also provided isa transgenic seed having in its genome any of the recombinant DNAconstructs presently disclosed, and a transgenic plant grown from suchtransgenic seed.

The transgenic plant cell or plant of the invention can be any suitableplant cell or plant of interest. Stably transformed transgenic plantsare particularly preferred. In many preferred embodiments, thetransgenic plant is a fertile transgenic plant from which seed can beharvested, and thus the invention further claims seed of such transgenicplants, wherein the seed is preferably also transgenic, that is,preferably contains the recombinant construct of the invention.

Where a recombinant DNA construct is used to produce a transgenic plantcell or transgenic plant of the invention, thee transformation caninclude any of the well-known and demonstrated methods and compositions.Suitable methods for plant transformation include virtually any methodby which DNA can be introduced into a cell, such as by direct deliveryof DNA (e. g., by PEG-mediated transformation of protoplasts, byelectroporation, by agitation with silicon carbide fibers, and byacceleration of DNA coated particles), by Agrobacterium-mediatedtransformation, by viral or other vectors, etc. One preferred method ofplant transformation is microprojectile bombardment, for example, asillustrated in U.S. Pat. Nos. 5,015,580 (soy), U.S. Pat. No. 5,550,318(maize), U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No.6,153,812(wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice)and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865(maize), all of which are incorporated by reference.

Another preferred method of plant transformation isAgrobacterium-mediated transformation. In one preferred embodiment ofthe invention, the transgenic plant cell of the invention is obtained bytransformation by means of Agrobacterium containing a binary Ti plasmidsystem, wherein the Agrobacterium carries a first Ti plasmid and asecond, chimeric plasmid containing at least one T-DNA border of awild-type Ti plasmid, a promoter functional in the transformed plantcell and operably linked to a gene suppression construct of theinvention. See, for example, U.S. Pat. No. 5,159,135; De Framond (1983)Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179,which are incorporated by reference. In such a binary system, thesmaller plasmid, containing the T-DNA border or borders, can beconveniently constructed and manipulated in a suitable alternative host,such as E. coli, and then transferred into Agrobacterium.

Detailed procedures for Agrobacterium-mediated transformation of plants,especially crop plants, include, for example, procedures disclosed inU.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat.Nos. 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soy); U.S. Pat. No.5,591,616 (maize); U.S. Pat. No. 5,981,840 (maize); U.S. Pat. No.5,463,174 (brassicas), all of which are incorporated by reference.Similar methods have been reported for, among others, peanut (Cheng etal. (1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987)Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan and Lemaux (1994)Plant Physiol., 104:37); rice (Toriyama et al. (1988) Bio/Technology,6:10; Zhang et al. (1988) Plant Cell Rep., 7:379; wheat (Vasil et al.(1992) Bio/Technology, 10:667; Becker et al. (1994) Plant J., 5:299),and alfalfa (Masoud et al. (1996) Transgen. Res., 5:313). See also U.S.Patent Application Publication 2003/0167537 A1, incorporated byreference, for a description of vectors, transformation methods, andproduction of transformed Arabidopsis thaliana plants wheretranscription factors are constitutively expressed by a CaMV35Spromoter. Transgenic plant cells and transgenic plants can also beobtained by transformation with other vectors, such as, but not limitedto, viral vectors (e. g., tobacco etch potyvirus (TEV), barley stripemosaic virus (BSMV), and the viruses referenced in Edwardson andChristie, “The Potyvirus Group: Monograph No. 16, 1991, Agric. Exp.Station, Univ. of Florida, which is incorporated by reference),plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) or any other suitable cloning vector, when usedwith an appropriate transformation protocol, e. g., bacterial infection(e.g., with Agrobacterium as described above), binary bacterialartificial chromosome constructs, direct delivery of DNA (e. g., viaPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, andmicroprojectile bombardment). It would be clear to one of skill in theart that various transformation methodologies can be used and modifiedfor production of stable transgenic plants from any number of plantspecies of interest. All of the above-described patents and publicationsdisclosing materials and methods for plant transformation areincorporated by reference in their entirety.

Transformation methods to provide transgenic plant cells and transgenicplants containing stably integrated recombinant DNA are preferablypracticed in tissue culture on media and in a controlled environment.“Media” refers to the numerous nutrient mixtures that are used to growcells in vitro, that is, outside of the intact living organism.Recipient cell targets include, but are not limited to, meristem cells,callus, immature embryos or parts of embryos, and gametic cells such asmicrospores, pollen, sperm, and egg cells. It is contemplated that anycell from which a fertile plant can be regenerated can be useful as arecipient cell for practice of the invention. Callus can be initiatedfrom various tissue sources, including, but not limited to, immatureembryos or parts of embryos, seedling apical meristems, microspores, andthe like. Those cells which are capable of proliferating as callus canserve as recipient cells for genetic transformation. Practicaltransformation methods and materials for making transgenic plants ofthis invention (e. g., various media and recipient target cells,transformation of immature embryos, and subsequent regeneration offertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos.6,194,636 and 6,232,526 and U.S. Application Publication 2004/0216189,which are incorporated by reference.

In general transformation practice, DNA is introduced into only a smallpercentage of target cells in any one transformation experiment. Markergenes are generally used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theantibiotics or herbicides to which a plant cell may be resistant can bea useful agent for selection. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells can be tested further to confirm stable integration of therecombinant DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin or paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(EPSPS). Examples of useful selective marker genes and selection agentsare illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and6,118,047, all of which are incorporated by reference. Screenablemarkers or reporters, such as markers that provide an ability tovisually identify transformants can also be employed. Non-limitingexamples of useful screenable markers include, for example, a geneexpressing a protein that produces a detectable color by acting on achromogenic substrate (e. g., beta-glucuronidase (GUS) (uidA) orluciferase (luc)) or that itself is detectable, such as greenfluorescent protein (GFP) (gfp) or an immunogenic molecule. Those ofskill in the art will recognize that many other useful markers orreporters are available for use.

Detecting or measuring the resulting change in expression of the targetgene (or concurrent expression of a gene of interest) obtained bytranscription of the recombinant construct in the transgenic plant ofthe invention can be achieved by any suitable methods, including proteindetection methods (e. g., western blots, ELISAs, and otherimmunochemical methods), measurements of enzymatic activity, or nucleicacid detection methods (e. g., Southern blots, northern blots, PCR,RT-PCR, fluorescent in situ hybridization). Such methods are well knownto those of ordinary skill in the art as evidenced by the numeroushandbooks available; see, for example, Joseph Sambrook and David W.Russell, “Molecular Cloning: A Laboratory Manual” (third edition), ColdSpring Harbor Laboratory Press, NY, 2001; Frederick M. Ausubel et al.(editors) “Short Protocols in Molecular Biology” (fifth edition), JohnWiley and Sons, 2002; John M. Walker (editor) “Protein ProtocolsHandbook” (second edition), Humana Press, 2002; and Leandro Peña(editor) “Transgenic Plants: Methods and Protocols”, Humana Press, 2004,which are incorporated by reference.

Other suitable methods for detecting or measuring the resulting changein expression of the target gene (or concurrent expression of a gene ofinterest) obtained by transcription of the recombinant DNA in thetransgenic plant of the invention include measurement of any other traitthat is a direct or proxy indication of expression of the target gene(or concurrent expression of a gene of interest) in the transgenic plantin which the recombinant DNA is transcribed, relative to one in whichthe recombinant DNA is not transcribed, e. g., gross or microscopicmorphological traits, growth rates, yield, reproductive or recruitmentrates, resistance to pests or pathogens, or resistance to biotic orabiotic stress (e. g., water deficit stress, salt stress, nutrientstress, heat or cold stress). Such methods can use direct measurementsof a phenotypic trait or proxy assays (e. g., in plants, these assaysinclude plant part assays such as leaf or root assays to determinetolerance of abiotic stress).

The recombinant DNA constructs of the invention can be stacked withother recombinant DNA for imparting additional traits (e. g., in thecase of transformed plants, traits including herbicide resistance, pestresistance, cold germination tolerance, water deficit tolerance, and thelike) for example, by expressing or suppressing other genes. Constructsfor coordinated decrease and increase of gene expression are disclosedin U.S. Patent Application Publication 2004/0126845 A1, incorporated byreference.

Seeds of transgenic, fertile plants can be harvested and used to growprogeny generations, including hybrid generations, of transgenic plantsof this invention that include the recombinant DNA construct in theirgenome. Thus, in addition to direct transformation of a plant with arecombinant DNA construct, transgenic plants of the invention can beprepared by crossing a first plant having the recombinant DNA with asecond plant lacking the construct. For example, the recombinant DNA canbe introduced into a plant line that is amenable to transformation toproduce a transgenic plant, which can be crossed with a second plantline to introgress the recombinant DNA into the resulting progeny. Atransgenic plant of the invention with one recombinant DNA (effectingchange in expression of a target gene) can be crossed with a plant linehaving other recombinant DNA that confers one or more additionaltrait(s) (such as, but not limited to, herbicide resistance, pest ordisease resistance, environmental stress resistance, modified nutrientcontent, and yield improvement) to produce progeny plants havingrecombinant DNA that confers both the desired target sequence expressionbehavior and the additional trait(s).

Typically, in such breeding for combining traits the transgenic plantdonating the additional trait is a male line and the transgenic plantcarrying the base traits is the female line. The progeny of this crosssegregate such that some of the plant will carry the DNA for bothparental traits and some will carry DNA for one parental trait; suchplants can be identified by markers associated with parental recombinantDNA Progeny plants carrying DNA for both parental traits can be crossedback into the female parent line multiple times, e. g., usually 6 to 8generations, to produce a progeny plant with substantially the samegenotype as one original transgenic parental line but for therecombinant DNA of the other transgenic parental line.

Yet another aspect of the invention is a transgenic plant grown from thetransgenic seed of the invention. This invention contemplates transgenicplants grown directly from transgenic seed containing the recombinantDNA as well as progeny generations of plants, including inbred or hybridplant lines, made by crossing a transgenic plant grown directly fromtransgenic seed to a second plant not grown from the same transgenicseed.

Crossing can include, for example, the following steps:

-   (a) plant seeds of the first parent plant (e. g., non-transgenic or    a transgenic) and a second parent plant that is transgenic according    to the invention;-   (b) grow the seeds of the first and second parent plants into plants    that bear flowers;-   (c) pollinate a flower from the first parent with pollen from the    second parent; and-   (d) harvest seeds produced on the parent plant bearing the    fertilized flower.

It is often desirable to introgress recombinant DNA into elitevarieties, e. g., by backcrossing, to transfer a specific desirabletrait from one source to an inbred or other plant that lacks that trait.This can be accomplished, for example, by first crossing a superiorinbred (“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrentparent), which carries the appropriate gene(s) for the trait inquestion, for example, a construct prepared in accordance with thecurrent invention. The progeny of this cross first are selected in theresultant progeny for the desired trait to be transferred from thenon-recurrent parent “B”, and then the selected progeny are mated backto the superior recurrent parent “A”. After five or more backcrossgenerations with selection for the desired trait, the progeny arehemizygous for loci controlling the characteristic being transferred,but are like the superior parent for most or almost all other genes. Thelast backcross generation would be selfed to give progeny which are purebreeding for the gene(s) being transferred, i. e., one or moretransformation events.

Through a series of breeding manipulations, a selected DNA construct canbe moved from one line into an entirely different line without the needfor further recombinant manipulation. One can thus produce inbred plantswhich are true breeding for one or more DNA constructs. By crossingdifferent inbred plants, one can produce a large number of differenthybrids with different combinations of DNA constructs. In this way,plants can be produced which have the desirable agronomic propertiesfrequently associated with hybrids (“hybrid vigor”), as well as thedesirable characteristics imparted by one or more DNA constructs.

Genetic markers can be used to assist in the introgression of one ormore DNA constructs of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers can provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers can be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized. The usefulness ofmarker assisted selection in breeding transgenic plants of the currentinvention, as well as types of useful molecular markers, such as but notlimited to SSRs and SNPs, are discussed in PCT Application PublicationWO 02/062129 and U.S. Patent Application Publications Nos. 2002/0133852,2003/0049612, and 2003/0005491, each of which is incorporated byreference in their entirety.

In certain transgenic plant cells and transgenic plants of theinvention, it may be desirable to concurrently express (or suppress) agene of interest while also regulating expression of a target gene.Thus, in some embodiments, the transgenic plant contains recombinant DNAfurther including a gene expression (or suppression) element forexpressing at least one gene of interest, and regulation of expressionof a target gene is preferably effected with concurrent expression (orsuppression) of the at least one gene of interest in the transgenicplant.

Thus, as described herein, the transgenic plant cells or transgenicplants of the invention can be obtained by use of any appropriatetransient or stable, integrative or non-integrative transformationmethod known in the art or presently disclosed. The recombinant DNAconstructs can be transcribed in any plant cell or tissue or in a wholeplant of any developmental stage. Transgenic plants can be derived fromany monocot or dicot plant, such as, but not limited to, plants ofcommercial or agricultural interest, such as crop plants (especiallycrop plants used for human food or animal feed), wood- or pulp-producingtrees, vegetable plants, fruit plants, and ornamental plants.Non-limiting examples of plants of interest include grain crop plants(such as wheat, oat, barley, maize, rye, triticale, rice, millet,sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such asforage grasses and forage dicots including alfalfa, vetch, clover, andthe like); oilseed crop plants (such as cotton, safflower, sunflower,soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (suchas walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane,coconut, date palm, olive, sugarbeet, tea, and coffee; wood- orpulp-producing trees; vegetable crop plants such as legumes (forexample, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus,artichoke, celery, carrot, radish, the brassicas (for example, cabbages,kales, mustards, and other leafy brassicas, broccoli, cauliflower,Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example,cucumbers, melons, summer squashes, winter squashes), edible alliums(for example, onions, garlic, leeks, shallots, chives), edible membersof the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers,groundcherries), and edible members of the Chenopodiaceae (for example,beet, chard, spinach, quinoa, amaranth); fruit crop plants such asapple, pear, citrus fruits (for example, orange, lime, lemon,grapefruit, and others), stone fruits (for example, apricot, peach,plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado,and berries; and ornamental plants including ornamental floweringplants, ornamental trees and shrubs, ornamental groundcovers, andornamental grasses. Preferred dicot plants include, but are not limitedto, canola, cotton, potato, quinoa, amaranth, buckwheat, safflower,soybean, sugarbeet, and sunflower, more preferably soybean, canola, andcotton. Preferred monocots include, but are not limited to, wheat, oat,barley, maize, rye, triticale, rice, ornamental and forage grasses,sorghum, millet, and sugarcane, more preferably maize, wheat, and rice.

The ultimate goal in plant transformation is to produce plants which areuseful to man. In this respect, transgenic plants of the invention canbe used for virtually any purpose deemed of value to the grower or tothe consumer. For example, one may wish to harvest the transgenic plantitself, or harvest transgenic seed of the transgenic plant for plantingpurposes, or products can be made from the transgenic plant or its seedsuch as oil, starch, ethanol or other fermentation products, animal feedor human food, pharmaceuticals, and various industrial products. Forexample, maize is used extensively in the food and feed industries, aswell as in industrial applications. Further discussion of the uses ofmaize can be found, for example, in U.S. Pat. Nos. 6,194,636, 6,207,879,6,232,526, 6,426,446, 6,429,357, 6,433,252, 6,437,217, and 6,583,338 andPCT Publications WO 95/06128 and WO 02/057471, each of which isincorporated by reference in its entirety.

Thus, in preferred embodiments, a transgenic plant of the invention hasat least one altered trait, relative to a plant lacking said recombinantDNA construct, selected from the group of traits consisting of:

-   (a) improved abiotic stress tolerance;-   (b) improved biotic stress tolerance;-   (c) improved resistance to a pest or pathogen of the plant;-   (d) modified primary metabolite composition;-   (e) modified secondary metabolite composition;-   (f) modified trace element, carotenoid, or vitamin composition;-   (g) improved yield;-   (h) improved ability to use nitrogen or other nutrients;-   (i) modified agronomic characteristics;-   (j) modified growth or reproductive characteristics; and-   (k) improved harvest, storage, or processing quality.

The invention further provides a method of providing at least onealtered plant tissue, including: (a) providing a transgenic plantincluding a regenerated plant prepared from a transgenic plant cellhaving in its genome any of the recombinant DNA constructs presentlydisclosed, or a progeny plant of the regenerated plant; and (b)transcribing the recombinant DNA construct in at least one tissue of thetransgenic plant, whereby an altered trait in the at least one tissueresults, relative to tissue wherein the recombinant DNA construct is nottranscribed, the altered trait being selected from:

-   (i) improved abiotic stress tolerance;-   (ii) improved biotic stress tolerance;-   (iii) improved resistance to a pest or pathogen of the plant;-   (iv) modified primary metabolite composition;-   (v) modified secondary metabolite composition;-   (vi) modified trace element, carotenoid, or vitamin composition;-   (vii) improved yield;-   (viii) improved ability to use nitrogen or other nutrients;-   (ix) modified agronomic characteristics;-   (x) modified growth or reproductive characteristics; and-   (xi) improved harvest, storage, or processing quality.    In preferred embodiments of the method of providing at least one    altered plant tissue, the transgenic plant from which such tissue is    obtained is a crop plant as described herein.

In particularly preferred embodiments, the transgenic plant ischaracterized by: improved tolerance of abiotic stress (e. g., toleranceof water deficit or drought, heat, cold, non-optimal nutrient or saltlevels, non-optimal light levels) or of biotic stress (e. g., crowding,allelopathy, or wounding); by improved resistance to a pest or pathogen(e. g., insect, nematode, fungal, bacterial, or viral pest or pathogen)of the plant; by a modified primary metabolite (e. g., fatty acid, oil,amino acid, protein, sugar, or carbohydrate) composition; a modifiedsecondary metabolite (e. g., alkaloids, terpenoids, polyketides,non-ribosomal peptides, and secondary metabolites of mixed biosyntheticorigin) composition; a modified trace element (e. g., iron, zinc),carotenoid (e. g., beta-carotene, lycopene, lutein, zeaxanthin, or othercarotenoids and xanthophylls), or vitamin (e. g., tocopherols)composition; improved yield (e. g., improved yield under non-stressconditions or improved yield under biotic or abiotic stress); improvedability to use nitrogen or other nutrients; modified agronomiccharacteristics (e. g., delayed ripening; delayed senescence; earlier orlater maturity; improved shade tolerance; improved resistance to root orstalk lodging; improved resistance to “green snap” of stems; modifiedphotoperiod response); modified growth or reproductive characteristics(e. g., intentional dwarfing; intentional male sterility, useful, e. g.,in improved hybridization procedures; improved vegetative growth rate;improved germination; improved male or female fertility); improvedharvest, storage, or processing quality (e. g., improved resistance topests during storage, improved resistance to breakage, improved appealto consumers); or any combination of these traits.

In one preferred embodiment, transgenic seed, or seed produced by thetransgenic plant, has modified primary metabolite (e. g., fatty acid,oil, amino acid, protein, sugar, or carbohydrate) composition, amodified secondary metabolite (e. g., alkaloids, terpenoids,polyketides, non-ribosomal peptides, and secondary metabolites of mixedbiosynthetic origin) composition, a modified trace element (e. g., iron,zinc), carotenoid (e. g., beta-carotene, lycopene, lutein, zeaxanthin,or other carotenoids and xanthophylls), or vitamin (e. g., tocopherols,) composition, an improved harvest, storage, or processing quality, or acombination of these. For example, it can be desirable to modify theamino acid (e. g., lysine, methionine, tryptophan, or total protein),oil (e. g., fatty acid composition or total oil), carbohydrate (e. g.,simple sugars or starches), trace element, carotenoid, or vitamincontent of seeds of crop plants (e. g., canola, cotton, safflower,soybean, sugarbeet, sunflower, wheat, maize, or rice), preferably incombination with improved seed harvest, storage, or processing quality,and thus provide improved seed for use in animal feeds or human foods.In another instance, it can be desirable to change levels of nativecomponents of the transgenic plant or seed of a transgenic plant, forexample, to decrease levels of proteins with low levels of lysine,methionine, or tryptophan, or to increase the levels of a desired aminoacid or fatty acid, or to decrease levels of an allergenic protein orglycoprotein (e. g., peanut allergens including ara h 1, wheat allergensincluding gliadins and glutenins, soy allergens including P34 allergen,globulins, glycinins, and conglycinins) or of a toxic metabolite (e. g.,cyanogenic glycosides in cassava, solanum alkaloids in members of theSolanaceae).

EXAMPLES Example 1

This example illustrates the construction and use of vectors designedfor double-stranded RNAi suppression or for anti-sense suppression of aluciferase gene. The gene suppression experiments used were similar to adual luciferase assay described by Horstmann et al. (2004) BMCBiotechnol., 4:13, which is incorporated by reference herein. A priorart vector, “vector 1A”, designed for double-stranded RNAi suppressionof a luciferase gene was constructed as depicted in FIG. 1A with an RNAitranscription unit with a polyadenylation site including (a) a chimericpromoter including an enhanced CaMV35S promoter linked to an enhancerelement (an intron from heat shock protein 70 of Zea mays, Pe35S-Hspintron), (b) an inverted repeat of DNA coding for firefly luciferase(LUC) with anti-sense oriented DNA followed by a sense oriented DNA, and(c) a 3′UTR DNA from Agrobacterium tumefaciens nopaline synthase gene(3′NOS) which provides a polyadenylation (polyA) site. Elements of theplasmid comprising the RNAi transcription unit had a DNA sequence of SEQID NO. 1. See Table 1 for a description of the elements within SEQ IDNO. 1. TABLE 1 Nucleotide position Element in SEQ ID NO. 1 CaMV e35Spromoter  1-614 Hsp 70 intron  645-1448 Firefly luciferase anti-sense1455-1025 Firefly luciferase sense 2082-2502 3′ UTR from nopalinesynthase 2515-2767

A prior art vector, “vector 1B”, designed for anti-sense suppression ofa luciferase gene and containing a polyA site was constructed asdepicted in FIG. 1B with an anti-sense transcription unit including (a)the CaMV e35S-Hsp 70 intron chimeric promoter as described in Table 1,(b) the firefly luciferase anti-sense sequence described in Table 2, and(c) the 3′ UTR from nopaline synthase as described in Table 1.

A novel vector, “vector 1C”, designed for double-stranded RNAisuppression of a luciferase gene was constructed as depicted in FIG. 1Cwith an RNAi transcription unit without a polyadenylation site andincluding (a) the CaMV e35S-Hsp 70 intron chimeric promoter as describedin Table 1, and (b) an inverted repeat of DNA coding for fireflyluciferase, including the firefly luciferase anti-sense and fireflyluciferase sense sequences described in Table 1. The RNAi transcriptionunit did not have 3′UTR DNA sequence providing a functionalpolyadenylation site.

Another novel vector, “vector 1D”, designed for anti-sense suppressionof a luciferase gene and without a functional polyadenylation site wasconstructed as depicted in FIG. 1D with an anti-sense transcription unitwithout polyadenylation site and including (a) the CaMV e35S-Hsp 70intron chimeric promoter and (b) the firefly luciferase anti-sensesequence described in Table 1. The RNAi transcription unit did not have3′UTR DNA sequence providing a functional polyadenylation site.

Maize protoplasts were prepared as previously described by Sheen (1990)Plant Cell, 2:1027-1038, which is incorporated by reference herein. Eachof the four vectors 1A through 1D was electroporated together withreporter vectors for firefly luciferase and Renilla luciferase intothree separate volumes of maize protoplasts. Two sets of fireflyluciferase suppression experiments were performed to confirm theenhanced ability for gene suppression exhibited by the constructswithout a functional polyadenylation site (vectors 1C and 1D) relativeto the anti-sense construct with a functional polyadenylation site(vector 1B). The relative level of suppression of the target gene,firefly luciferase, was indicated by the ratio of firefly luciferase toRenilla luciferase “ffLUC/rLUC”, and the results of the two experimentsare given in Table 2. TABLE 2 Average ffLUC/rLUC First Second VectorDescription of Construct experiment experiment 1A RNAi with polyA site1862 2387 1B anti-sense with polyA site 6089 13988 1C RNAi without polyAsite 3620 5879 1D anti-sense without polyA site 2238 4762

Example 2

This example further illustrates the construction and use of vectorsdesigned for double-stranded RNAi suppression or for anti-sensesuppression of a luciferase gene. The gene suppression experiments usedwere similar to a dual luciferase assay described by Horstmann et al.(2004) BMC Biotechnol., 4:13. The vectors illustrated in FIG. 2 wereconstructed. Vector 2A (FIG. 2A), a control vector not encodinganti-sense or double-stranded RNA for the target gene (fireflyluciferase), consisted of (a) the CaMV e35S-Hsp 70 intron chimericpromoter as described in Example 1 and Table 1, (b) an inverted repeatof DNA coding for beta-glucuronidase (GUS) (uidA) with anti-senseoriented DNA followed by a sense oriented DNA, and (c) a 3′UTR DNA fromAgrobacterium tumefaciens nopaline synthase gene (3′NOS) as described inExample 1 and Table 1, which provides a polyadenylation (polyA) site.Vector 2B (FIG. 2B), a prior art vector designed for double-strandedRNAi suppression of a luciferase gene, consisted of (a) the CaMVe35S-Hsp 70 intron chimeric promoter as described in Example 1 and Table1, (b) an inverted repeat of DNA coding for firefly luciferase (LUC)with anti-sense oriented DNA followed by a sense oriented DNA, asdescribed in Example 1 and Table 1, and (c) a 3′UTR DNA fromAgrobacterium tumefaciens nopaline synthase gene (3′NOS) as described inExample 1 and Table 1, which provides a polyadenylation (polyA) site.Vector 2C (FIG. 2C), a novel vector, consisted of (a) the CaMV e35S-Hsp70 intron chimeric promoter as described in Example 1 and Table 1, (b)the firefly luciferase anti-sense sequence, as described in Example 1and Table 1, (c) spacer DNA consisting of a 3′UTR DNA from Agrobacteriumtumefaciens nopaline synthase gene (3′NOS) as described in Example 1 andTable 1, and (d) the firefly luciferase sense sequence, as described inExample 1 and Table 1. Vector 2D (FIG. 2D), a novel vector, consisted of(a) the CaMV e35S-Hsp 70 intron chimeric promoter as described inExample 1 and Table 1, (b) a first copy of the firefly luciferaseanti-sense sequence, as described in Example 1 and Table 1, (c) spacerDNA consisting of a 3′UTR DNA from Agrobacterium tumefaciens nopalinesynthase gene (3′NOS) as described in Example 1 and Table 1, and (d) asecond copy of the firefly luciferase anti-sense sequence. Vector 2E(FIG. 2E), a prior art vector designed for anti-sense RNA suppression ofa luciferase gene, consisted of (a) the CaMV e35S-Hsp 70 intron chimericpromoter as described in Example 1 and Table 1, (b) the fireflyluciferase anti-sense sequence, as described in Example 1 and Table 1,and (c) a 3′UTR DNA from Agrobacterium tumefaciens nopaline synthasegene (3′NOS) as described in Example 1 and Table 1, which provides apolyadenylation (polyA) site.

Each of the four vectors was electroporated together with reportervectors for firefly luciferase and Renilla luciferase into threeseparate volumes of maize protoplasts prepared as previously describedby Sheen (1990) Plant Cell, 2:1027-1038. Firefly luciferase suppressionexperiments were performed, and the relative level of suppression of thetarget gene, firefly luciferase, was indicated by the logarithm of theratio of firefly luciferase to Renilla luciferase, “log(Fluc/Rluc)”, asdepicted in FIG. 3.

Example 3

This example describes transformation of a crop plant (maize) with anenhanced anti-sense construct. A plasmid for binary vectorAgrobacterium-mediated transformation of maize is constructed includingthe elements shown in FIG. 4. Specifically, the plasmid includes annptII gene as an antibiotic selectable marker and a recombinant DNAconstruct for enhanced anti-sense gene suppression, consisting of aCaMV35S promoter operably linked to transcribable DNA consisting ofabout 300 base pairs of a green fluorescent protein (gfp) gene in ananti-sense orientation, wherein a functional polyadenylation site isabsent in this transcribable DNA. The plasmid also includes left T-DNAborder (LB) and right T-DNA border (RB) elements. A control plasmid forRNAi suppression of green fluorescent protein (GFP) is constructed byadding to the enhanced anti-sense construct shown in FIG. 4 a repeat ofthe gfp DNA in the sense orientation followed by a 3′ NOS elementincluding a functional polyadenylation site. Maize callus fortransformation is selected from a transgenic maize line expressing GFP.Both the plasmid with the enhanced anti-sense construct and the controlplasmid with the RNAi construct are inserted into maize callus byAgrobacterium-mediated transformation. Events are selected as beingresistant to kanamycin. The efficiency of suppression with enhancedanti-sense constructs is substantially the same as with the RNAiconstructs.

Example 4

This example illustrates the use of a recombinant DNA construct fornon-systemic suppression of a target gene in specific tissue of atransgenic plant. Specifically, this example describes transformation ofa crop plant (maize) with an enhanced anti-sense construct. A plasmidfor binary vector Agrobacterium-mediated transformation of corn isconstructed including the elements shown in FIG. 5A. Specifically, theplasmid includes an aroA gene as an herbicidal selectable marker and arecombinant DNA construct for enhanced anti-sense gene suppression,consisting of a seed-specific maize L3 oleosin promoter (as disclosed inU.S. Pat. No. 6,433,252, incorporated herein by reference) operablylinked to transcribable DNA consisting of about 300 base pairs of theLKR domain of a maize lysine ketoglutarate reductase/saccharopinedehydrogenase gene (LKR/SDH) in an anti-sense orientation, wherein afunctional polyadenylation site is absent in this transcribable DNA. Theplasmid also includes left T-DNA border (LB) and right T-DNA border (RB)elements. The plasmid with the enhanced anti-sense construct is insertedinto maize callus by Agrobacterium-mediated transformation. Events areselected as being resistance to glyphosate herbicide and grown intotransgenic maize plants to produce F1 seed. Mature seeds from each eventare analyzed to determine success of transformation and suppression ofLKR/SDH. The mature transgenic seeds are dissected to extract proteinfor Western analysis. Seed from transgenic maize plants shows reductionin LKR/SDH and increased lysine as compared to wild type.

In a further development of this approach, a recombinant DNA constructof the present invention is constructed as follows. A plasmid for binaryvector Agrobacterium-mediated transformation of corn is constructed asshown in FIG. 5B, which includes a recombinant DNA construct of thepresent invention for gene suppression, including left and right T-DNAborders containing between them a promoter element operably linked to anintron (maize heat shock protein 70 intron, I-Zm-hsp70) within which isembedded a first gene suppression element for suppressing at least onefirst target gene (in this non-limiting example, the at least one firsttarget gene includes coding sequence from the LKR domain, codingsequence from the SDH domain, or non-coding sequence of the maize lysineketoglutarate reductase/saccharopine dehydrogenase gene (LKR/SDH), orany combination of these). The first gene suppression element caninclude any gene suppression element as described above under theheading “Gene Suppression Elements” wherein the intron is locatedadjacent to the promoter element. In the specific, non-limitingembodiment depicted in FIG. 5B, the promoter element is anendosperm-specific maize B32 promoter (nucleotides 848 through 1259 ofGenBank accession number X70153, see also Hartings et al. (1990) PlantMol. Biol., 14:1031-1040, which is incorporated herein by reference),although other promoter elements could be used. This specific embodimentalso includes an aroA gene as an herbicidal selectable marker; otherselectable marker or reporter genes can be used, e. g., a selectablemarker conferring glyphosate resistance, epsps-cp4(5-enolpyruvylshikimate-3-phosphate synthase from Agrobacteriumtumefaciens strain CP4). The intron-embedded gene suppression elementincludes any one or more gene suppression elements, including, forexample, single or multiple copies of sense or anti-sense, tandem orinterrupted repeats, single or multiple sense/anti-sense pairs able toform dsRNA for gene suppression, gene suppression sequences derived froman miRNA, sequences including siRNAs, or combinations of any of these.The construct optionally includes a gene expression element (e. g.,transcribable or translatable DNA outside of the intron), a second genesuppression element, or both.

In one non-limiting example, the gene suppression element includes anabout 300 base-pair anti-sense DNA segment that is anti-sense to thetarget gene, maize lysine ketoglutarate reductase/saccharopinedehydrogenase gene (LKR/SDH), wherein a functional polyadenylation siteis absent in this transcribable heterologous DNA. The plasmid alsoincludes left T-DNA border (LB) and right T-DNA border (RB) elements.The plasmid with the intron-embedded transcribable heterologous DNA isinserted into maize callus by Agrobacterium-mediated transformation.Events are selected as being resistance to glyphosate herbicide andgrown into transgenic maize plants to produce F1 seed. Mature seeds fromeach event are analyzed to determine success of transformation andsuppression of LKR/SDH. The mature transgenic seeds are dissected toextract protein for Western analysis. Seed from transgenic maize plantsshows endosperm-specific reduction in LKR/SDH and increased lysine ascompared to wild type.

Example 5

This example illustrates use of recombinant DNA constructs for pestcontrol in plants producing by means of gene suppression in a specifictissue of a transgenic plant. Specifically, this example describestransformation of a crop plant (soybean) with an enhanced anti-senseconstruct. A plasmid for binary vector Agrobacterium-mediatedtransformation of soybean is constructed including the elements shown inFIG. 6. Specifically, the plasmid includes an aroA gene as an herbicidalselectable marker and a recombinant DNA construct for enhancedanti-sense gene suppression, consisting of a TUB-1 root specificpromoter from Arabidopsis thaliana (disclosed in FIG. 1 of U.S. PatentApplication Publication 2004/078841 A1, incorporated by referenceherein) operably linked to transcribable DNA consisting of anti-senseoriented DNA of a nematode major sperm protein (msp) of a soybean cystnematode (disclosed as SEQ ID NO:5 in U.S. Patent ApplicationPublication 2004/0098761 A1, incorporated herein by reference), whereina functional polyadenylation site is absent in this transcribable DNA.The plasmid also includes left T-DNA border (LB) and right T-DNA border(RB) elements. The plasmid with the enhanced anti-sense construct isinserted into soybean callus by Agrobacterium-mediated transformation.Events are selected as being resistance to glyphosate herbicide.Reduction in soybean cyst nematode infestation as compared to wild typeis observed.

In a further development of this approach, a recombinant DNA constructof the present invention is constructed as follows. A plasmid for binaryvector Agrobacterium-mediated transformation of corn is constructed,which includes an aroA gene as an herbicidal selectable marker and arecombinant DNA construct of the present invention for gene suppression,consisting of a TUB-1 root specific promoter from Arabidopsis thaliana(disclosed in FIG. 1 of U.S. Patent Application Publication 2004/078841A1, incorporated by reference herein) operably linked to an intron(maize alcohol dehydrogenase intron, I-Zm-adh1) within which is embeddeda first gene suppression element for suppression of an endogenous geneof a crop plant pest (soybean cyst nematode); in this specific,non-limiting example, the gene suppression element is transcribableheterologous DNA that includes an anti-sense DNA segment that isanti-sense to the target gene, nematode major sperm protein of a soybeancyst nematode (disclosed as SEQ ID NO:5 in U.S. Patent ApplicationPublication 2004/0098761 A1, incorporated herein by reference), whereinthe resulting transcribed RNA is unpolyadenylated. As a selectablemarker, the plasmid alternatively uses a gene conferring glyphosateresistance, epsps-cp4 (5-enolpyruvylshikimate-3-phosphate synthase fromAgrobacterium tumefaciens strain CP4). Other promoters, firsttranscribable heterologous DNAs, or introns can be substituted; theconstruct optionally includes a gene expression element, a secondtranscribable heterologous DNA for suppressing a second target gene, orboth. The plasmid optionally contains a transcribable or translatablegene expression element outside of the intron. The plasmid also includesleft T-DNA border (LB) and right T-DNA border (RB) elements. The plasmidwith the enhanced anti-sense construct is inserted into soybean callusby Agrobacterium-mediated transformation. Events are selected as beingresistance to glyphosate herbicide. Reduction in soybean cyst nematodeinfestation as compared to wild type is observed.

Example 6

This example illustrates a recombinant DNA construct of the invention,specifically, a construct including a gene suppression element thatcontains intron-embedded tandem repeats. More specifically, thisillustrates a construct including a suppression element that containsintron-embedded tandem repeats for suppression of at least one targetmicroRNA precursor. The tandem repeats are designed to suppress at leastone target sequence selected from said at least one target microRNAprecursor or a promoter of said at least one target microRNA precursoror both. This example also describes methods for testing recombinant DNAconstructs for their ability to silence a target gene, and optionallyfor their ability to concurrently express a gene of interest.

Gene silencing by tandem repeats may operate through a nuclear-localizedheterochromatin-associated RNAi pathway. See, for example, Sijen et al.(1996) Plant Cell, 8:2277-2294, Ma and Mitra (2002) Plant J., 31:37-49,Zilberman et al. (2003) Science, 299:716-719, and Martienssen (2003)Nat. Genet., 35:213-214, which are incorporated by reference herein. Thepresent invention provides recombinant DNA constructs for enhancednuclear-localized gene silencing (e. g., suppression of production ofmature microRNA). Non-limiting examples of such constructs areconstructs with one or more suppression elements including tandemrepeats, where the tandem repeats are embedded in an intron; suchconstructs can optionally include a gene expression element (FIG. 7A),which can be upstream (5′, not shown) or downstream (3′, as shown) ofthe intron. Two other approaches for enhancing nuclear-localized genesilencing by tandem repeats are tandem repeats that are transcribed butnot processed for transport into the cytoplasm, e. g., transcribed fromconstructs lacking a functional terminator, as shown in FIG. 7B, andtandem repeats under transcriptional control of two opposing promoters,as shown in FIG. 7C. By embedding tandem repeats in an intron (e. g.,FIG. 7A), transgenic transcripts splice out the tandem repeat containingintron in the nucleus. By removing or omitting a functional terminatorof a transgene cassette (e. g., FIG. 7B), the resulting RNA transcriptscontaining tandem repeats are without a polyA signal and more likely toaccumulate in the nucleus. In a construct where tandem repeats areflanked by opposing or convergent promoters (e. g., FIG. 7C), onepromoter will transcribe the sense strand, and the other will transcribethe antisense strand; these two complementary strands can form a dsRNA.The purpose for this is to provide the initial dsRNA substrate for aDicer or a Dicer-like enzyme. Thus, for example, Dicer produces siRNAs,and RDR2-dependent amplification of dsRNA and siRNAs, facilitated by thetandem repeat configuration, maintains the silencing pathway for thesesequences.

In the non-limiting examples shown in FIG. 7, there are two copies inthe tandem repeat. Also encompassed by the invention are embodimentswith the copy number of the tandem repeat ranging from 2 to about 100,as well as embodiments with tandem or interrupted repeats of one or moresequences (in non-limiting examples, these could include, e. g., AABB,AABAA, AABAABAA, AABAABB, ABBBBAA, AAABBB, AABBAA, and otherarrangements, where A and B represent discrete sequences, each of whichcan be repeated). The size of each repeat is preferably at least about19, or at least about 21, or at least about 50, at least about 100, atleast about 200, or at least about 500 nucleotides in length.Preferably, at least two of the repeats are in the tandem repeatorientation. Unique or non-repeated sequences, including repeats of asecond sequence, can optionally occur as “spacers” between some or allof the repeated units. Such spacers are preferably at least about 4, atleast about 10, or at least about 20 nucleotides in length. Havingunique sequences between facilitate assembly and/or verification of thetandem repeats. The repeats can be arranged in either the sense orantisense orientation, or, for example, where there are repeats of morethan one sequence, each sequence can independently be in an arrangementof tandem sense repeats or tandem anti-sense repeats.

Example 7

This example describes non-limiting methods for testing any of therecombinant DNA constructs of the invention for their ability to silencea target gene, and optionally for their ability to concurrently expressa gene of interest. Constructs can be designed and tested in transientassays by various means known to one skilled in the art, for example,protoplast transient transformation and Agrobacterium infiltrationassays. For example, constructs can be designed where the target gene isa gene easily assayed for suppression (e. g., green fluorescent proteinor GFP, luciferase or luc, or other reporter or marker genes commonlyused). Such transient assays can generally be used to test anyrecombinant DNA constructs, e. g., constructs containing intron-embeddedgene suppression elements (including gene suppression elements otherthan tandem repeats) for their ability to suppress a target gene.

In one non-limiting example, experiments to assay for gene suppressionof a target gene (the reporter gene, luciferase) are carried out with amaize protoplast model system. Maize protoplasts are prepared aspreviously described by Sheen (1990) Plant Cell, 2:1027-1038, which isincorporated by reference herein. Polyethylene glycol (PEG)-mediatedtransformations (see, for example, Armstrong et al. (1990), Plant CellRep., 9:335-339, which is incorporated by reference herein) areperformed in deep well (2 milliliters/well) 96-well plates. Separatevectors containing either firefly luciferase or Renilla luciferase areemployed as reporters. The firefly luciferase reporter vector includes achimeric promoter including a chimeric promoter including an enhancedcauliflower mosaic virus (CaMV) 35S promoter linked to an enhancerelement (an intron from heat shock protein 70 of Zea mays), the codingsequence of the firefly luciferase gene luc, and a 3′untranslated region(3′ UTR) DNA from Agrobacterium tumefaciens nopaline synthase gene(3′NOS) which provides a polyadenylation (polyA) site. The Renillaluciferase reporter vector includes the same chimeric promoter, thecoding sequence of the Renilla luciferase gene luc, and the same 3′NOSUTR terminator. Generally, 1.3 micrograms firefly luciferase reportervector DNA, 0.6 micrograms Renilla luciferase reporter vector DNA, andadditional plasmid (pUC18) DNA are added to each well in order tomaintain the total amount of RNA plus DNA constant at 12.5 microgramsper well. To each well is added 160 microliters (2×10⁶ protoplasts permilliliter) of maize protoplasts. Protoplasts are madetransformation-competent by treatment with a solution containing 4 gramsPEG 4000, 2 milliliters water, 3 milliliters 0.8 molar mannitol, and 1milliliter Ca(NO₃)₂. The protoplasts are co-transformed with the testrecombinant DNA constructs of the invention, where the target gene isfirefly luciferase, together with the reporter vectors for fireflyluciferase and Renilla luciferase, into 4 separate volumes of maizeprotoplasts; the test constructs can be delivered in a vector. Therelative level of suppression of the target gene, firefly luciferase, isindicated by the intensity of firefly luciferase emission (“Fluc”)normalized to Renilla luciferase emission (Rluc). A negative controltest vector is, for example, one similar to the test vectors containingthe gene suppression elements but containing a gene suppression elementtargetting a non-relevant gene such as beta-glucuronidase (GUS) (uidA).A positive control test vector is, for example, one similar to the testvector but containing, for example, the full-length firefly luc gene.The relative level of suppression of the target gene, fireflyluciferase, is given as the logarithm of the ratio of firefly luciferaseemission to Renilla luciferase emission, “log(Fluc/Rluc)”.

Transient assays such as the one described in the preceding paragraphcan be designed to optionally simultaneously assay for expression of agene of interest. For example, a model gene of interest can include GFP.The experiments are carried out as in the preceding paragraph, where thetest recombinant DNA constructs can contain both a first genesuppression element for suppressing the target gene and a geneexpression element for expressing a gene of interest such as GFP. Theexpression of GFP can be simultaneously monitored by spectrophotometryas is the firefly and Renilla luciferase emission.

Example 8

This example describes various non-limiting embodiments of recombinantDNA constructs of the invention and useful in making transgeniceukaryotes (including transgenic plant cells, plants, and seeds) of theinvention. One non-limiting application of these constructs is, forexample, suppression of at least one target miRNA precursor or miRNApromoter, or non-systemic gene suppression of a gene endogenous to aplant or to a pest or pathogen of the plant.

FIG. 8A schematically depicts non-limiting examples of recombinant DNAconstructs of the invention for suppression of at least one target gene.These constructs include at least one first gene suppression element(“GSE” or “GSE1”) for suppressing at least one first target gene,wherein the first gene suppression element is embedded in an intronflanked on one or on both sides by non-protein-coding DNA. Theseconstructs utilize an intron (in many embodiments, an intron derivedfrom a 5′ untranslated region or an expression-enhancing intron ispreferred) to deliver a gene suppression element without requiring thepresence of any protein-coding exons (coding sequence). The constructscan optionally include at least one second gene suppression element(“GSE2”) for suppressing at least one second target gene, at least onegene expression element (“GEE”) for expressing at least one gene ofinterest (which can be coding or non-coding sequence or both), or both.In embodiments containing an optional gene expression element, the geneexpression element can be located outside of (e. g., adjacent to) theintron. In some embodiments, the intron containing the first genesuppression element is 3′ to a terminator.

To more clearly differentiate recombinant DNA constructs of theinvention (containing at least one gene suppression element embeddedwithin a single intron flanked on one or on both sides bynon-protein-coding DNA) from the prior art, FIG. 8B schematicallydepicts examples of prior art recombinant DNA constructs. Theseconstructs can contain a gene suppression element that is locatedadjacent to an intron flanked by protein-coding sequence, or between twodiscrete introns (wherein the gene suppression element is not embeddedin either of the two discrete introns), or can include a gene expressionelement including a gene suppression element embedded within an intronwhich is flanked by multiple exons (e. g., exons including the codingsequence of a protein).

Example 9

This example describes various non-limiting embodiments of genesuppression constructs of the invention. FIG. 9 depicts variousnon-limiting examples of gene suppression elements and transcribableexogenous DNAs useful in the recombinant DNA constructs of theinvention. Where drawn as a single strand (FIGS. 9A through 9E), theseare conventionally depicted in 5′ to 3′ (left to right) transcriptionaldirection; the arrows indicate anti-sense sequence (arrowhead pointingto the left), or sense sequence (arrowhead pointing to the right). Thesegene suppression elements and transcribable exogenous DNAs can include:DNA that includes at least one anti-sense DNA segment that is anti-senseto at least one segment of the at least one first target gene, or DNAthat includes multiple copies of at least one anti-sense DNA segmentthat is anti-sense to at least one segment of the at least one firsttarget gene (FIG. 9A); DNA that includes at least one sense DNA segmentthat is at least one segment of the at least one first target gene, orDNA that includes multiple copies of at least one sense DNA segment thatis at least one segment of the at least one first target gene (FIG. 9B);DNA that transcribes to RNA for suppressing the at least one firsttarget gene by forming double-stranded RNA and includes at least oneanti-sense DNA segment that is anti-sense to at least one segment of theat least one target gene and at least one sense DNA segment that is atleast one segment of the at least one first target gene (FIG. 9C); DNAthat transcribes to RNA for suppressing the at least one first targetgene by forming a single double-stranded RNA and includes multipleserial anti-sense DNA segments that are anti-sense to at least onesegment of the at least one first target gene and multiple serial senseDNA segments that are at least one segment of the at least one firsttarget gene (FIG. 9D); DNA that transcribes to RNA for suppressing theat least one first target gene by forming multiple double strands of RNAand includes multiple anti-sense DNA segments that are anti-sense to atleast one segment of the at least one first target gene and multiplesense DNA segments that are at least one segment of the at least onefirst target gene, and wherein said multiple anti-sense DNA segments andthe multiple sense DNA segments are arranged in a series of invertedrepeats (FIG. 9E); and DNA that includes nucleotides derived from amiRNA, or DNA that includes nucleotides of a siRNA (FIG. 9F).

FIG. 9F depicts various non-limiting arrangements of double-stranded RNAthat can be transcribed from embodiments of the gene suppressionelements and transcribable exogenous DNAs useful in the recombinant DNAconstructs of the invention. When such double-stranded RNA is formed, itcan suppress one or more target genes, and can form a singledouble-stranded RNA or multiple double strands of RNA, or a singledouble-stranded RNA “stem” or multiple “stems”. Where multipledouble-stranded RNA “stems” are formed, they can be arranged in“hammerheads” or “cloverleaf” arrangements.

Example 10

This example describes various non-limiting embodiments of recombinantDNA constructs of the invention and useful in making transgeniceukaryotes (including transgenic plant cells, plants, and seeds) of theinvention. More specifically, this example describes embodiments of genesuppression constructs that transcribe to RNA capable of formingmultiple double-stranded “stems” and suppress one or more target genes.

To form a double “hairpin” molecule or a double-stranded RNA structureresembling a “hammerhead”, a recombinant DNA construct is designed toinclude a single-stranded, contiguous DNA sequence including twonon-identical pairs of self-complementary sequences is used, wherein theDNA can transcribe to RNA also including two non-identical pairs ofself-complementary sequences that can form two separate double-strandedRNA “stems”. Each member of a non-identical pair of self-complementarysequences preferably includes at least about 19 to about 27 nucleotides(for example 19, 20, 21, 22, 23, or 24 nucleotides) for every targetgene that the recombinant DNA construct is intended to suppress; in manyembodiments the pair of self-complementary sequence can be larger thanat least about 19 to about 27 base pairs (for example, more than about30, about 50, about 100, about 200, about 300, about 500, about 1000,about 1500, about 2000, about 3000, about 4000, or about 5000 basepairs) for every target gene that the recombinant DNA construct isintended to suppress. Each non-identical pair of self-complementarysequences can be separated by spacer DNA, for example, additionalnucleotides that can form a loop connecting the two strands of RNAforming a double-stranded hairpin, or that can separate adjacentdouble-stranded RNA “stems”. Spacer DNA can include nucleotides that arelocated at the distal end of one or both members of the pair theself-complementary sequences, for example, where inclusion of thesenucleotides as “spacer” sequence facilitates the formation of thedouble-stranded RNA structures, or facilitates the assembly andmaintenance of these sequences in plasmids. Spacer DNA can includesequence encoding an aptamer. The non-identical pair ofself-complementary sequences can include sequence derived from a singlesegment of a single target gene, multiple copies of a single segment ofa single target gene, multiple segments of a single target gene,segments of multiple target genes, or any combination of these, with orwithout spacer DNA. Multiple “hairpins” can be formed in an analogousfashion by including more than two non-identical pairs ofself-complementary sequences that can form two separate double-strandedRNA “stems”.

A specific, non-limiting example of this configuration of sequences isshown in FIG. 10, which depicts a gene suppression element (“GSE”, FIG.10A) useful in recombinant DNA constructs of the invention, and arepresentation of the type of RNA double hairpin molecule that it wouldbe expected to produce (FIG. 10B). The double hairpin molecule isdepicted with a 3′ untranslated region including a polyadenylated tail;however, embodiments of the invention also include analogous constructsthat produce a double hairpin molecule lacking a polyadenylated tail ora 3′ untranslated region. In this example, orientations of the sequencesare anti-sense followed by sense for sequence 1, then sense followed byanti-sense for sequence 2 (FIG. 10A). This arrangement may beconvenient, e. g., when both sequence 1 and 2 are derived from the sametarget gene, in which cases the sense sequences can represent sequencesthat are contiguous in the native target gene. However, any order ofsense and anti-sense sequences can be used in the recombinant DNAconstruct, as long as the transcribed RNA is capable of forming twoseparate double-stranded RNA “stems”. Analogous recombinant DNAconstructs could be designed to provide RNA molecules containing morethan 2 double-stranded “stems”, as shown in FIG. 10C, which depicts anRNA molecule containing 3 “stems”.

Example 11

This example describes a non-limiting embodiment of the recombinant DNAconstruct of the invention, and methods for its use. More particularly,this example describes a recombinant DNA construct containing a genesuppression construct that transcribes to RNA capable of formingmultiple double-stranded “stems” and that suppresses a first targetgene, wherein the recombinant DNA construct can be transcribed in atransgenic plant, and the first target gene is a gene native to a pestor pathogen of the transgenic plant.

In this non-limiting example, an RNA molecule that is capable ofgenerating a double hairpin structure is designed to be transcribed froma recombinant DNA construct containing a gene suppression elementsimilar to that shown in FIG. 10A. In this specific example, the genesuppression element (“GSE”) contains a first sense sequence and secondsense sequence (as depicted in FIG. 10A), which are contiguous sequencesfrom SEQ ID NO. 2 (a 872 nucleotide segment of the cDNA sequence of thecorn root worm vacuolar ATPase gene). However, this method can be usedfor noncontiguous sequences, including sequences from different genes.The complete gene suppression element given as SEQ ID NO. 3 contains DNAsequences of SEQ ID NO. 2 arranged as follows: the reverse complement ofthe DNA segment starting at nucleotide 1 and ending at nucleotide 300 ofSEQ ID NO. 2, followed by the DNA segment starting at nucleotide 100 andending at nucleotide 600 of SEQ ID NO. 2, followed by the reversecomplement of the DNA segment staring at nucleotide 300 and ending atnucleotide 500 of SEQ ID NO. 2. This gene suppression element (SEQ IDNO. 3) is embedded in a suitable intron (as described above under theheading “Introns”) that is operably linked to a suitable promoterelement (as described above under the heading “Promoter Elements”).Where it is desirable to transcribe RNA that is transported out of thenucleus, a terminator element can be included either embedded in theintron containing the GSE and operably linked to (5′ to) the genesuppression element, or outside of and 5′ to the intron containing theGSE.

Example 12

This example describes a non-limiting embodiment of the recombinant DNAconstruct of the invention, and methods for its use. More particularly,this example describes a recombinant DNA construct containing a genesuppression construct that suppresses a miRNA precursor molecule, e. g.,a pri-miRNA.

The primary transcript of a miRNA gene (MIR gene), termed a pri-miRNA,is believed to be hundreds to thousands nucleotides in length andlargely processed in the nucleus to a smaller (generally less than 100nucleotides) stem-loop structure, which is then exported to thecytoplasm for further processing into a mature miRNA. By embedding agene suppression element for suppressing a miRNA precursor molecule (forexample, DNA that transcribes to RNA for suppressing a pri-miRNA byforming double-stranded RNA, preferably double-stranded RNA that lackspolyadenylation) into a spliceable intron, the resulting double-strandedRNA is expected to remain in the nucleus due to the absence ofcis-acting nuclear export signals, resulting in suppression of the miRNAthat is more efficient than that achieved by constructs that producecytoplasmic dsRNA. Another potential advantage of this approach is thatthe miRNA precursors offer larger target sequences for suppression thandoes a mature miRNA. Alternatively, an intron-embedded gene suppressionelement can be designed to target the promoter sequences of the miRNAprecursor, resulting in transcriptional gene silencing. See, forexample, Matzke and Birchler (2005) Nat. Rev. Genet., 6:24-35, Matzke etal. (2004), Biochim. Biophys. Acta, 1677:129-141, and Papp et al. (2003)Plant Physiol., 132:1382-1390, all of which are incorporated byreference herein.

One general, non-limiting design for a recombinant DNA constructincludes a suppression element for suppressing production of a maturemiRNA and preferably designed to target the pri-miRNA sequence of atargeted MIR gene, wherein the gene suppression element is embedded inan intron (e. g., a heat shock 70, actin 1, or alcohol dehydrogenaseintron) flanked on one or on both sides by non-protein-coding DNA, whichis fused to a reporter gene (e. g., beta-glucuronidase GUS, or greenfluorescent protein GFP) and driven by a constitutive (e. g., 35S) ortissue specific (e. g., B32) promoter. Such a construct generallyresembles that shown in FIG. 7A, where the reporter gene can be upstreamor downstream of the intron. The recombinant DNA construct can betransformed into Arabidopsis by standard techniques. Expression of theoptional reporter gene confirms the proper processing of the intron inthe transgenic Arabidopsis in which the construct is transcribed.

In a non-limiting specific example of this approach, a recombinant DNAconstruct of the invention is designed to suppress a specific allele,MIR164c, of the Arabidopsis thaliana microRNA gene MIR164.Loss-of-function of this allele, eep1, caused by T-DNA insertion, hasbeen shown to increase the number of petals of early flowers inArabidopsis (see Baker et al. (2005) Curr. Biol., 15:303-315, which isincorporated by reference herein). One specific, non-limiting constructincludes a heat shock 70 intron, within which is embedded a suppressionelement including DNA that transcribes to a sense/anti-sensedouble-stranded RNA for suppressing the pri-miRNA of MIR164c sequence,fused to GFP and driven by a 35S promoter. GFP expression confirmstranscription and proper splicing of the construct in Arabidopsis plantstransformed with the construct. The “early extra petal” phenotype ofeep1 is used to score for the miRNA164c suppression.

In another non-limiting, specific example of this approach, arecombinant DNA construct of the invention is designed to suppress theArabidopsis thaliana microRNA gene MIR172, which regulates the mRNA of afloral homeotic gene, APETALA2 (X. Chen (2004) Science, 303:2022-2025).Elevated miRNA172 accumulation results in floral organ identity defectssimilar to those in loss-of-function apetala2 mutants. On the otherhand, the expression of mutant APETALA2 mRNA resistant to miRNA172causes different floral patterning defects. One specific,non-limiting construct includes a heat shock 70 intron, within which isembedded a suppression element (for example, DNA that transcribes to asense/anti-sense double-stranded RNA for suppressing the pri-miRNA ofMIR162 sequence), fused to GFP and driven by a 35S promoter. GFPexpression confirms transcription and proper splicing of the constructin Arabidopsis plants transformed with the construct. The floralpatterning defect phenotype is used to score for the miRNA172suppression.

Example 13

This example describes a non-limiting embodiment of a recombinant DNAconstruct of the invention, and methods for its use. More particularly,this example describes identifying a MIR gene in maize and, further,making and using a recombinant DNA construct containing a genesuppression element that suppresses production of the mature miRNAtranscribed from the identified MIR gene in maize.

A single small RNA was isolated and cloned using procedures based onpublished protocols (Llave et al. (2002) Plant Cell, 14:1605-1619, andLau et al. (2001) Science, 294:858-862). Low molecular weight RNA wasisolated from developing maize endosperm. Adaptors were ligated followedby RT-PCR for conversion of RNA to DNA. Additional PCR amplificationfollowed by TA cloning and sequencing led to the identification of ahighly abundant 22-mer in maize endosperm corresponding to the DNAsequence TGAAGCTGCCAGCATGATCTGG (SEQ ID NO. 4). Sequence alignmentanalysis showed that the isolated 22-mer sequence is homologous to arice sequence annotated as “Oryza sativa precursor microRNA 167g gene,complete sequence” (GenBank accession number AY551238, gi:45593912) andhaving the sequence,

GAAGATATTAGTTCTTGCTGGTGTGAGAGGCTGAAGCTGCCAGCATGATCTGGTCCATGAGTTGCACTGCTGAATATATTGAATTCAGCCAGGAGCTGCTACTGCAGTTCTGATCTCGATCTGCATTCGTTGTTCTGAGCTATGTATGGATTTGATCGGTTTGAAGGCATCCATGTCTTTAATTTCATCGATCAGATCATGTTGCAGCTTCACTCTCTCACTACCAGCAAAACCATCTCA (SEQ ID NO. 5, with the homologousnucleotides indicated by bold, underlined text). A proprietary maizegenomic DNA sequence database was searched for sequences containing22-mer segments identical to SEQ ID NO. 4 or to its complement. Thesequences thus identified included overlapping SEQ ID NO. 6, SEQ ID NO.7, and SEQ ID NO. 8, as given in Table 3, with the location of the22-mer indicated by underlined text. TABLE 3 SEQ ID NO. Sequence 6GTTTTGGCTTGTTCACCCCTCATGTGCACATGCTGTTACTCCGAAGCTTGCGCTTTTGTATTCGTTGTTGCATTGCAACCATCCCCGCCGAAGGTGAGCCGAAGGTAATCTTGGGTATTCTACCTGCAACACTTATTATTCAAGCTACAAAACAGTTGTCGAGTTAGTTTTTTTTTTACCTTCGAAAAGAAGACTTCCGGCAATGCACAACTTCCCATCTGCATTATCGTGAGCAGGATTGTAGGCACACAGTGATGACGAAGACAGAGACAGCAATATACACAACCGAACCAAGAGAGAAGCAAAGGCATAATAATAAAAAAAGAGAGAGGAAACTAGATCGACAAGGCCATTATTATCACGGATAATTAATCAACGTCGTCAACGGCGGAAATAAGCTAGCTTGACTGGTGGTCTCTGGCGAGTGCAGCATGGATATGAATTGCAGGAGGGTGAGCTAGCTAGGGTTTTCGATGTGCGGCCACCAGCAGATGAAACTACAGCATGACCTGGTCCTGGTGCTCATTAATTACCCTCTCTCTCTCTCCCTTCCCCTCTCATCTTGGATTCGTCGATCCATATATGACAGTCAGGGACGGGGGAGAGAGAGAGAGTGACAGGGGCCGGTAGTAGTATAGATTACATCCATTTTACATATACCACCACCATCATAA CCAGATCATGCTGGCAGCTTCA CCAACTCGTGGTGCACCACTACATACCCTCTCGTCTGATCCAAACGGAGGAAG GAGGAAGAA 7TTGGCTTGTTCACCCCTCATGTGCACATGCTGTTACTCCGAAGCTTGCGCTTTTGTATTCGTTGTTGCATTGCAACCATCCCCGCCGAAGGTGAGCCGAAGGTAATCTTGGGTATTCTACCTGCAACACTTATTAATTCAAGCTACAAAACAGTTGTCGAGTTAGTTTTTTTTTTACCTTCGAAAAGAAGACTTCCGGCAATGCACAACTTCCCATCTGCATTATCGTGAGCAGGATTGTAGGCACACAGTGATGACGAAGACAGAGACAGCAATATACACAACCGAACCAAGAGAGAAGCAAAGGCATAATAATAAAAAAAGAGAGAGGAAACTAGATCGACAAGGCCATTATTATCACGGATAATTAATCAACGTCGTCAACGGCGGAAATAAGCTAGCTTGACTGGTGGTCTCTGGCGAGTGCAGCATGGATATGAATTGCAGGAGGGTGAGCTAGCTAGGGTTTTCGATGTGCGGCCACCAGCAGATGAAACTACAGCATGACCTGGTCCTGGTGCTCATTAATTACCCTCTCTCTCTCTCCCTTCCCCTCTCATCTTGGATTCGTCGATCCATATATGACAGTCAGGGACGGGGGAGAGAGAGAGAGTGACAGGGGCCGGTAGTAGTATAGATTACATCCATCTTACATATACCACCACCATCATAA CCAGATCATGCTGGCAGCTTCA CCAACTCGTGGTGCACCACTACATACCCTCTCGTCTGATCCAAACGGAGGAAGGAGGAAGAAGAGCTAGCTATCCGAGAGAGAGGGAGAGGGTAGAGAGATGGAGAGAGCGAGGAATGAATTGAAGAACCGAGGGATAGCTATAGCTATATATATATGGGGATGGGGAGGCCAACGTCTCGCTCACTCGC 8TATTCTACCTGCAACACTTATTAATTCAAGCTACAAAACAGTTGTCGAGTTAGTTTTTTTTTTACCTTCGAAAAGAAGACTTCCGGCAATGCACAACTTCCCATCTGCATTATCGTGAGCAGGATTGTAGGCACACAGTGATGACGAAGACAGAGACAGCAATATACACAACCGAACCAAGAGAGAAGCAAAGGCATAATAATAAAAAAAGAGAGAGGAAACTAGATCGACAAGGCCATTATTATCACGGATAATTAATCAACGTCGTCAACGGCGGAAATAAGCTAGCTTGACTGGTGGTCTCTGGCGAGTGCAGCATGGATATGAATTGCAGGAGGGTGAGCTAGCTAGGGTTTTCGATGTGCGGCCACCAGCAGATGAAACTACAGCATGACCTGGTCCTGGTGCTCATAATTACCCTCTCTCTCTCTCCCTTCCCCTCTCATCTTGGATTCGTCGATCCATATATGACAGTCAGGGACGGGGGAGAGAGAGAGAGTGACAGGGGCCGGTAGTAGTATAGATTACATCCATCTTACATATACCACC ACCATCATAACCAGATCATGCTGGCAGCTTCA CCAACTCGTGGTGCACCACTACATACCCTCTCGTCTGATCCAAACGGAGGAAGGAGGAAGAAGAGCTAGCTATCCGAGAGAGAGGGAGAGGGTAGAGAGATGGAGAGAGCGAGGAATGAATTGAAGAACCGAGGGATAGCTATAGCTATATATATATGGGATGGGGAGGCCAACGTCTCGCTCACTCGCAGCGTATTTTGATGCCCTTTTTTATTTGTTGCATTTCGATCCATTTTCTTTTGTCCTGCGCTTTTTTCGTACGATGTTTGTTGCAAGGATAAGCCTTTCGG

These three sequences (SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8)overlapped to give a single contiguous sequence SEQ ID NO. 9,

GTTTTGGCTTGTTCACCCCTCATGTGCACATGCTGTTACTCCGAAGCTTGCGCTTTTGTATTCGTTGTTGCATTGCAACCATCCCCGCCGAAGGTGAGCCGAAGGTAATCTTGGGTATTCTACCTGCAACACTTATTAATTCAAGCTACAAAACAGTTGTCGAGTTAGTTTTTTTTTTACCTTCGAAAAGAAGACTTCCGGCAATGCACAACTTCCCATCTGCATTATCGTGAGCAGGATTGTAGGCACACAGTGATGACGAAGACAGAGACAGCAATATACACAACCGAACCAAGAGAGAAGCAAAGGCATAATAATAAAAAAAGAGAGAGGAAACTAGATCGACAAGGCCATTATTATCACGGATAATTAATCAACGTCGTCAACGGCGGAAATAAGCTAGCTTGACTGGTGGTCTCTGGCGAGTGCAGCATGGATATGAATTGCAGGAGGGTGAGCTAGCTAGGGTTTTCGATGTGCGGCCACCAGCAGATGAAACTACAGCATGACCTGGTCCTGGTGCTCATTAATTACCCTCTCTCTCTCTCCCTTCCCCTCTCATCTTGGATTCGTCGATCCATATATGACAGTCAGGGACGGGGGAGAGAGAGAGAGTGACAGGGGCCGGTAGTAGTATAGATTACATCCATCTTACATATACCACCACCATCATAACCAGATCATGCTGGCAGCTTCACCAACTCGTGGTGCACCACTACATACCCTCTCGTCTGATCCAAACGGAGGAAGGAGGAAGAAGAGCTAGCTATCCGAGAGAGAGGGAGAGGGTAGAGAGATGGAGAGAGCGAGGAATGAATTGAAGAACCGAGGGATAGCTATAGCTATATATATATGGGGATGGGGAGGCCAACGTCTCGCTCACTCGCAGCGTATTTTGATGCCCTTTTTTATTTGTTGCATTTCGATCCATTTTCTTTTGTCCTGCGCTTTTTTCGTACGATGTTTGTTGCAAGGATAAGCCTTTCGG (with the location of the 22-mer indicated by bold,underlined text). This was identified as a maize MIR167 sequence whichtranscribes to a pri-miRNA. Recombinant DNA constructs of the invention,containing one or more suppression elements for suppressing theidentified MIR167 pri-miRNA (or a pre-miRNA) are designed andtransformed into maize plants by procedures such as those describedabove under the heading “Recombinant DNA Constructs for SuppressingProduction of Mature miRNA and Methods of Use Thereof” and elsewhere inthis disclosure (e. g., by Agrobacterium-mediated transformation). Onenon-limiting suppression element is an inverted repeat containing one ormore sense and anti-sense pairs of SEQ ID NO. 4, embedded in an intron.Suppression of production of the mature miRNA corresponding to theidentified MIR gene is detected by analysis of low molecular weight RNAfrom resulting transgenic maize endosperm and other tissues (e. g.,embryo, leaf, root, flower) for example, by using a labelled oligoprobecorresponding to the 22-mer (SEQ ID NO. 4 or its complement). Transgenicsuppression of production of the mature miRNA encoded by a MIR167 gene,is useful, for example, for identifying related genetic elements or tomanipulate the pathways that are controlled by MIR167, e. g., byidentifying target genes suppressed by a mature miRNA encoded by aMIR167 gene. Thus, the transgenic tissues are also analyzed formorphological and compositional changes (such as, but not limited to,changes in primary metabolite, secondary metabolite, trace element,carotenoid, or vitamin composition or modified responses to biotic orabiotic stress, or modified yield) to assess the function of the maizeMIR167.

Example 14

This example describes novel mature miRNAs and MIR genes identified incrop plants (maize and soy). Novel MIR sequences were identified inproprietary expressed sequence tag (EST) sequence databases from cropplants. The criteria that were used for identifying MIR genes included aconserved miRNA sequence of at least 19 nucleotides, a stable predictedfold-back structure encompassing the miRNA in one arm, and the absenceof a significant open reading frame (ORF). Seven MIR sequences wereidentified in maize (Zea mays): SEQ ID NO. 10 (Zm-MIR164e, including theDNA sequence SEQ ID NO. 11 corresponding to the conserved mature miRNAmiR164e), SEQ ID NO. 12 (Zm-MIR319-like, including the DNA sequence SEQID NO. 13 corresponding to the conserved mature miRNA miR319-like), SEQID NO. 14 (Zm-MIR393b, including the DNA sequence SEQ ID NO. 15corresponding to the conserved mature miRNA miR393b), SEQ ID NO. 16(Zm-MIR399g, including the DNA sequence SEQ ID NO. 17 corresponding tothe conserved mature miRNA miR399g), SEQ ID NO. 18 (Zm-MIR408b,including the DNA sequence SEQ ID NO. 19 corresponding to the conservedmature miRNA miR408b), SEQ ID NO. 20 (Zm-MIR398, including the DNAsequence SEQ ID NO. 21 corresponding to the conserved mature miRNAmiR398), and SEQ ID NO. 22 (Zm-MIR397, including the DNA sequence SEQ IDNO. 23 corresponding to the conserved mature miRNA miR397). Six MIRsequences were identified in soybean (Glycine max): SEQ ID NO. 24(Gm-MIR393a, including the DNA sequence SEQ ID NO. 25 corresponding tothe conserved mature miRNA miR393a), SEQ ID NO. 26 (Gm-MIR393b,including the DNA sequence SEQ ID NO. 27 corresponding to the conservedmature miRNA miR393b), SEQ ID NO. 28 (Gm-MIR399, including the DNAsequence SEQ ID NO. 29 corresponding to the conserved mature miRNAmiR399), SEQ ID NO. 30 (Gm-MIR164a, including the DNA sequence SEQ IDNO. 31 corresponding to the conserved mature miRNA miR164a), SEQ ID NO.32 (Gm-MIR164b, including the DNA sequence SEQ ID NO. 33 correspondingto the conserved mature miRNA miR164b), and SEQ ID NO. 34 (Gm-MIR164c,including the DNA sequence SEQ ID NO. 35 corresponding to the conservedmature miRNA miR164c). The novel MIR sequences are given in Table 4,with the location of nucleotides corresponding to the mature miRNAindicated by underlined text. TABLE 4 SEQ ID NO. Sequence 10GTATGTTCTCCGCTCACTCCCCCATTCCACTCTCATCCATCTCTCAAGCTACACACATATAAAAAAAAAAGAGTAGAGAAGGACCGCCGTTAGAGCACTTGATGCATGCGTACGTCGATCCGGCGGACCGATCTGCTTTTGCTTGTGTGCTTGGTGAGAAGGTCCCTGTTGGAGAAGCAGGGCACGTGCAG AGACACGCCGGAGCACGGCCGCCGCCGATCTACCGACCTCCCACACCTGCCTTGTGGTGTGGGGGTGGAGGTCNNNNNNCGNAGCGAGAGCTGNCGNTGNTGNTTNGATGCTGNTNGCTCCTCCTGCNCGTGCTCCCCTTCTCCACCACGGCCTTCTCACCACCCTCCTCCCCCGGCGGCGGCGGCGGCGGACCGCCCTTGCCGCGATCAATAATGAAACCAAAAGCCGACAGTGTTTGAGCAGGAAACACAAAAGGCGGATATCCCACTGNTAGCACTTCTGCGTTGATCATGGTCATCTGGAACAAAATAATACTTGGGGACTTTACAGCGAGTGCAGCATGCTTAAGCTAGTTC 12TTCGGTCCAAGTAGTGGTGGTCATAATATGCTCCAAATAAAAGAAAGGTGGAGGAGCATCTCACAGACGACACAGCTGCTATGCTAGCACACGTCGAATCAATAGCTAGTTGCATGCAAAGTTCCAAAGCAAATAAACAGTGAGATCGAAAGACGTTTCGCTGTTGCACGACACGACGAATCGATCGAACGAAAGTGTGTTTTTATGATTCCACAGATTCTCGTTTATATATAATCCTAGCTAGCTAATCTAGAACGTACAGTGCACACCATCTTCTTCCACAGATCACAGAAAGACAGCAGAAACCTGCATGGATCGGATCCGGTCCTGTCCTGTAAGATCTACACACATGCAAAGCAAATCAATTTCTTCCTTTTCTTTTCTTCAGAAACTGGGATAACTTTTTGGAAGAGATCGAACAGTATATAGATTCAGGGAGCAGATCAAGGATTATATATATAGCTAGTATGTGTACATATCAAAAGGGCAAGAAAAGTACAAAAAAGCATCGGATCTCCATTATATATACAACAGCTATATAACAACCACAGAAGAACAGTAAGCACGCACATGGTAAAATTAAAATAGCCTGGCAGCTGCTATGGATGTATGCATCAGATGCCTAATATATATGCAAGATAATAATTAATAAGCAGCTCAAGCAAAGACAGATCAAGAGTTCGAGACAGCAGGTTGGAAAATAAAATACAGATCATATGAAGTAAAACCTTGACTTGAGATACGAATGATGAAGCTGCATGGGTAAAGTAAACAAGGAAAGGATCGGAGGGAGCACCCTTCAGTCCAAGCAAAGACGGTGCGAGATCGAAGCTTTTACCTCCCGCTTCATTCACTCATCTGCGAAGCTCGTTTCCATGGCCGTTTGCTTGGCATGTGGGTGAATGAGTCGGCAGCTAATCCGACCCTAGCACCGCCCCTGA GTGGACTGAAGGACGCT CTCTTCCATCCGGCCGGCGACCATCGATCACAACCATGACGCCGCGCCCGGCGGCAAATATATTAACAAGAAATGAAATCAAAAGAGAGAGGAAGAACAAACATGATGCGCAGCTGCGCTAGCTAGTGCTTGATCTGTCTGACCACCTCATGGCGCGCAGTGTTTAGTTTTCTCCCTGGATCTTGCGAAGAAGGCGATGGATTTTCGATGGTTGCAAGGAGGAGCGACCGACAAAGGGTTTATATAATATGTAGACGGC 14GCCGGCCGGGTCGGGATGCCGCCTACTAGCAGGAAGCTAGTGGAGGAC TCCAAAG GGATCGCATTGATCTAACCTGCCGATCGACGCCGACGTACGTACGTGCCCGAGGACAAGCAGATCAGTCAGTGCAATCCCTTTGGAATTCTCCACTTAGCGCCTCCATCCCCGCGCCGCCCTCCAGGTTTCGCTTCGATCCATCCATGTTTCCTTCGTTTAAATTAGTTCGTTTGTTTTTTTTTTATTATTTATTTGATTCGCCGCCGCCGGTCTATCTACTCTGTTTGCAACGCCTTTCGATCCATCGGCTTCTATGTATGCTATAATTAAGGGTTTTTTTACATTGGTCCGATGCATGAGAGGAGCTGTGCAGACCAACATGGCAACCAATTACATCGATCTTGAGGACTCTTATGGACCAACATGCCAAGTTCTTCATTGCTTGTACTACCATTCAAGTTGTCAAACAATTACCAAATTAACTCAAGTATTCGAGAGAAGCATATATGTTAGTCAAATAGCAAATTCTTTACTAACTGATCTATGTACCGACATGTCAACTTCTTGCATACCAACGTGGCAAGAAGGTAATCATTGTTCATGAATAAGATTATCACTA 16CTAGGAATGGTACGGTGCTGGCTAAGCTAGCTAGATCATCGTCCTGGAGCTGAGAGCAGCAGCTACCTATATATCTAGCTGGTTTTCTAACGACGATGACGAACGACCGCGGGACTAGCATGATGCAGCTAGCTGAAGACAGTTGTAGGCAGCTCTCCTCTGGCAGGCAGGCGCGCGGTCATCGTCGCCATCGACGACGGTTGCTTGGCTCTGCTATGCTGTGTTCGTTCGGCCATGGTGTGCTAGCTAGCCGTGCATGCGTTGCAGTGTAACATGCGTGCATGCACGCGCGTACGTCC TGCCAAAGGAGAGTTGCCCTG CGACTGTCTTCAGCTCGAACAAGATCGACCGGCCCGGACAGGAATGTTGGGCGTACGTTGTCATCAGGGTTTAAGCTCCACGATTCCAAATATTCACCACTTCTGGGAGGAGTTTTGAAGCTGCTCGAAAGCATATTGTGTCTGAGTGTAATAAATCGGCGGGGAATCATATGTTCATGTTCTCACTGCAAGAATAAGCTTGTCAAAGAGGGTGGTGAAGTAAAATCTCACCTGATCAGCGGCACAGGTGCTCCTAGCGACGGGTGTAAGTCATGGAGGACAAGCAACAGGAAGTCCACTGCCAAGTGCTTCCATCGTCGTCAAATCACAGGTCAGGGGTTAATTATATGGGGGAAGAGGCCATTATCATCAGGTACGCGTGGTTCTCACACAGTCGGGGCCACGTTCGTTGATGATCTGCCTCTTCGGCATCTCAAACTCTTGTTGTGCTCTCTACATCAGTAGAGAAGGTGTGTTCACAAGTCGTTTCTTCTTAAGACTATGTTTTGGTTGATCTTGATCTATAGAACTATTTTATTGTAGAACTACTGAACCCTTTCGAAGTGTTGTACTCAATTTGTGTAGAACAATGCATGATTAATTTCTACCAATAGTCTACGGTAGCCGGTAGTTGTTTTATCCTACTAGAGAAATTGTTGCATGGTTAATTGGTTAATTTGTGTAGGATGTGCCAAAAGAAGAGGAAGAGAACACCATCAATATGAATGGTGAATTATTCGTAAGCTTATCTTCCACTAATGGTGCTGGAAGCCAGAAGGAGAAAGAGGAGGATGGAGATCATGTGTCAAGGCTCAGGAGATAAATCGAGGAAGAAAAAGATCGAAGGGTGGTGTTTAGTTGTATCCTTCCAAGTTCCAAGTTCACGGTAAAGAGAGGAAAGTGTGCTAGTTCAAGAGAGTATGGGATGGAGATAGGCACCATTGGACTTGGAGTGGAGGACAAGATGTTACCATTTTGCATTTCCATGGAGCGTGGAGACTTCTGAGTGCTTCAATCTTTTTATTAAAAATCAGTCTGAGCGATGATGAGTCTAAAGAGACTAAGACTATATCATAATCTACGATGGATTTAATCTATAAGGTGGATATATCACATATGGTTGCCAATCTTGTATATTTCATATTTGCATGGTTGGTAGTTGCACTGTTGCAATCTTAAGACCTGTATAGTTGCATATTTGATTGTGTTTTTAGAATGTTGATTTGTGGTTGTGC TCGCTTCTTTCT18 GGTACCTTTAGCGTTAGCACAGACACACACAGGTAAGGAGAGCGAGAGGTGGGTTGGGTTTGATCGGAGACAGGGACGAGGCAGAGCATGGGTAGGGGGCCATCAACAGAATTCCAAATTTGATTTCTGTTTGCTCGCTCACAAAATGGAGGGACTCACCACAAACACACTCAGGCGTTGTTGCTCCCTCCCC TGCACTGCCTCTTCCCTGGC TCCTCACCGTCTCCCATCCACCTATCCTCTCTCCTTTCTCTCTCGTTATGGTTTTGTATAATTTTTTTCCTGCATTCTTTTCTCAGTACAAGTCCTACACTAATTTGGCTGTCTTTGCACCAGTACTAATAAACACCGCAGGTCCCTGCAATAGGGTTTACAACAATTCTATTGTAATGACTGCTGTAAAACATCCGCATCATTTAATTCAACTTTCCGGTTTCAGTCAGCCCTGCAAAAGTGCTCCTCCGTTCGTCCGCGTTTGGTGTTGGCTTCTGCGGCTCCGGTGCCCAGAGTTGCTGCCGGCGGAGGCCGAGCAGGAGCGCAACTAACAAGAGCGGCCAAGGCGCCAGTGATCCTCACCATGGACAGGAGATCGATGGAGATGAGCGTGAGCTTCCGATGCTTCGGTACCCGAAGAAAAGAACGGGAACAAAGGCGAGAAACATGATCCACCTCTATGCTTTTTTGGCAACATATCCTATGCTTAAACAGTTATGGTGTTCAAATGTACACATTAATAGAGCGTTTGGTTTGAAGAATCACACCATCTAAAGAGGTGGTGCATCATGAATTTATTCCTTAAAAAAAAAAAAAAAAAAAAAAAA 20 GCCGGCCGGGTCGGGTGTGTTCTCAGGTCGCCCCCG ATCACAGCCAACGCGGGCGACCGCGCGCCATTATAGCACACGGGGCACGGCACGCCTTCGGCCTCCCACTAACTGCACAAGAGGACGACGCGGCAGCGAGGAGGGAGCAAAGGAAAGGGGATATGTCGAGGCCGCCCAACAGGAGCGACGCGCACCTCTCCGCCGAGGACGAGGCGGCGCTGGAGGCCGAGGTGCGGGAGTACTACGACGACGCGGCGCCAAAGCGCCACACCAAGCCCTCCCGCAGCGAGCACTCCGCCGTGTACGTCGACGCGCTCGTCCCGGACGTCGGCGGCAACTCCCACCCGGAGCTGGACAAGTTCCAAGAGCTGGAAGCCCACACCGAGAGGTTGGTGTACGAGGGCGCCAATGTGGGAGATGAGTTCGTAGAGACGGAGTACTACAAGGACCTCGGCGGCGTCGGCGAGCAGCACCACACGACCGGAACGGGCTTCATCAAGATGGACAAAGCTAAAGGCGCCCCCTTCAAACTGTCTGAAGATCCCAATGCAGAGGAGCGACATGCTTCTTGCAGGGGAAACCCTGCTACCAACGAGTGGATCCCGTCAGCTGACACGGTAAGACTGGGGGAGCACAGTCCAGTTTATCCTATGCAGGTGCAGGGTCGGCTCCAATCGGCGTCTCTACTGACGAACGCATCGTTAGCTTGTACCCAGCGTCAGACAAGCCAAGCAGAAGCGACAGCTGAGGGACTGTATATCTCAAGCCATGAGAATTCAGACGAGTGCTTTCCGCCATTAGAATAAGGAACCACACTGGTTGTCCACCGTATCTTCACTGTTCTGCGTCGAGATTCTTGTGATTCTTACGTGGAACAAATTAAGCGTGCTACGAGTTAGACCTCTGTGTTCTGGCTGTAAATGGCAAGGAATGAAGTTCTAATCGTGGTTCAGCAGTCAATCAATTACTGTGTTTCTGATCCTAAGGCTCTAGAAACAATCGGACCTTCAAAATAAACTAGGCGAAAATTCTATG TCGTTTCG 22GAGCGGGGTCTTGAAACTGGCTGCGCAGAAGGAAGGGATGAAGGGGTTCCTGGAGCTCGACGCCGAGGTTTTCGAGCTTGCCCCTTCGTTCTTTCTGGTCGAGCTGAAGAAGGCCAGCGGTGACACCATTGAGTACCAAAGGCTCGTGAGGGAAGAAGTGCGGCCTGCGCTGAAGGATATGGTCTGGGCTTGGCAGAGCGACCGGCACCAGCAGCAGGCAGCAGCGGTGCGAGCAGTCTGTGCAAGGAGAGGACCAGCAGCAGCCGTTGTCGTCTTTGCCGACGCAGCAGTAGTCACTGCACCACCAGTTGCGACCGCCATAACCAGATCACGTCAAAACTGCACCAAGCCGCACAGGACTAGTAACTCCCACTTGCATCGACGCTTATGTGATTGCGGAATTGTGTTTCAGGTTACCTGCCTGCTGCGGTAGGACCTAAAACGCCTACCTGCCTACCATTTGGCATTTTTTTGTATACTGTACGTACATTAGAGTAATAAACAAACATGCTTAACTTTTCAGCTTTCGATTGGAATGTGCTTTTCGATGTAACTCTGTAACCAGTGTAGGTACGAAGTCGATTAGCCACAGGGTCTGGCCATGTTGACCTCACGTAGCCCTGGTTCATTGGTGTAACAGTTTGTTGGCTGCGGCTTTACATTATTTTGTCTCTATGGATTACGGCTGCGACTATGTGTAGCTGAACAAGCTGGTTATGATGAGCCCTGGAAACGTGTGTTTACTGCAGCTATTTGCAGCCAGTGACTGTTGATACAAACGACGAAGTAGAGTTGGTTGTTTATGTAGGCACGCAGCATGACCATAATTATCCATGAATCATGGATAGATGCACAATGTTTAGGAAACAGGTGTGTGTGGCTGGCTGGTGGTGCGAGAAGAGATGCGCTGCCTTGATGTACTGTACTGGGACTGGGAGGGATGCGTCTCGCAGTACAGTCTGTACTATCATCTCTACACGCACGCACGCAGGCTCGACGTGTCGGCGGCGGCGGTCCAGACTCCATATGGATCCGTAGTAGTACAACCTGTTGGCGGGTAGTACAGGTTGGAGCACGCCTCTTCTTCAGTCTTCCTTCCTGAGATGAGGAGTCACTCACCAGGAAAACGCTTGCAGTACACCCCGCTCGCGGGCGTTGTTTATAGTGATCGGTAGCGTGAGCACAGAGCGCCATCAGAAGATGCAAAGAGA AAGAGAAGCAAAGGCATCATTGAGCGCAGCGTTGATG AGCCAGCCGCCGTGCCTCCCCTGTCGGCTGCGGCGGCTCACCAGCGCTGCACTCAATTACGCCTTTGCTTTCTCCCGCTGGCCGCGTGTGTGCAGAGCGGGCGGGCGTTCGGCATCATTCATCAGGTTTGCTTCATTTATTATGCACTCATCGAAGGCTTCTCCTTCGACACTGTCTAGGTGGCGCAGGATCTGAATCAGATGGGTGTCGTCTTCTTCCTCCATCTGCACTCCTGCCCCGTATGATGTCGGTGTCCTAGGACGGCCAGTTGTCTGCGTTCTGGTTAACCCAATTACCTGACGGGGCGGACGACGCTGATAATGATCAGAGAGAGCATGAGGCCATATGCAAGCCTAGACCTAGCTCCCAAACTATTAAAGGTTGCTTCGAGCCCTGGCTGTCATATCAACTACCAACCAGTTTATGTCGATTATCAGTTCCTATCTATCACAACGCTCCACTGTCCCCCTCTTTCGTAGACTTTTAGTTAACATCAAACAATGCATTTTATTGAAATCCAAAATACATCTGACTGCGTAATTGAGTAGATTTATCCCAAAATTTAATTAGCATGCCGCTGTGAGCTAGGAGAGCGACACTAGTTTACAATATGACAGTGTTTGTGTTCGGCCAAACCATTTTTGTTGATGGGTAAGGGGACACGACCCCCAAATAGACGCTCTCATTTTAATGAAGAATTAGTTGTGGACTAATTGATAATTCCCATTACAATCGGATTGCACGCATTAAATCTTAGTGCTAAGGAGGTGTTACAAATGAACCTAAAAAAGAAAAGATAATTGTTGAWTTATGTGGGTCTGGTCCATATTAATATTCAATAATTGTCAATGCTAGTTGTCACTTTATGCTACGGTGTACTAGTACTTACCAAACTAGAAGTTTAAGGGACAATTCACTYAACTTAAATAGGTGGACTATTGGTGCATCTATTGAGAAGCTGAGAAAAGGATGAAGGACTGTCACGCGTGCGCGCACCCTGATCTGTTGAGAAGCTGAGATCGTAGGAACAAGAATCACTAAATTCGGAGTTACAGATTTCAAGTTATGATTTTTCGAAGGTTTTATGTGTTTGGTACGGAATTGATTAAGTGATCAATTTTAATATGGGTTTCATGCTAAAACTGAGGTACTAAGTGGTAAACAAAATTATAGAAATTGGAATGGGTTAAAAAGGAGTTTGCATGATTTTCCTATGAATTATACAAGATTATGGATTTATTTTAATACCAAAATCACTTTTTATATTTATTTTACCCTGGTTTTCTATCCACTAGACTGCGCCCAAGATTATACTAAAGTTTAGGGGCAACTGCATAAAAAAACTAAGACTTAGGGCCCGTTTGTGATGGACTGCGGGTTGATAACTTAGAAACAGAGGGTCTCTTATGTAAACTGTATGTGCTGAAGGGGTATGAAGCATCTACGATCGTCAGATTACAATTCCACGGCCAGATTAAATCGCCAGTGCGATGAACCGTTACGTAACAGCCATCATCCGATCTGAGATCTACGACCCTGATTCTAAATGCCCTAAAACCTCCCAGATCCACTCCCTTTGTCCGAATCGGTACGCATCGGATTAAATCGCAGCCGCACTCTGATGGATCTACGGCCCACGCAGATCATCCCCCATACCAACGGCGAACGGGCGCCGCCGCCCGTAAACACGGCGGTGGCCATGGCCGTGGATGGCCAACTCGACTTCGAGGCCGTAATCCTCTAGTCTAAGACGTGCTACGTGGTAAGTGGATGAAGACGATTTCCATGGGTTCAGTACTTACCGAGGGCAAGGTCGTGCACAAGCTGTTCACGGCGAAGCGCGGCCGTAGCAAAAATTGAAAGGGAAATGTGACTTTGGGCTATTTCTATAAATGTTTTGGTGATTAGATGCCCAACACATATTGTTTTAGTTCATATGTGCTAAGTGATTGAGAAGTGCAAATCAAGAATCAAGGTATATTTCTAGCCCTAGTAAATTTCTTTTGGATACTAACATATCTCTCTAAGTGCTAGGGACACTACCAAGAAAAGTGGAAATGAACTGGAGAAGTTTGGCAGAGT 24ACCATTACACTCTTAGTGAATATTTCATAAAATATAAAGTTCCTCCTGGGCGAGAAACATCTCCATGTTTAAGGAAACAGTGCGAAGAATTATTACACCAGACATATTCAAGGCAACTAGTGGAATCCAATAAGGAATGCTGGCCCACTGCGGAAAATATTTCGGGTTGAATGATAGGGAAGGGGGTCATTCAACAAAAATCTTAATTTTCTCGGAGATTGGCAAATCTACATTGACAAGATAAATAAATAATTTATGAAAACAATAAAAAAATGATAATGGAAACAGGGCTTATAATATAAGCACTACTAAGCTAGTTTGTTTCTCCTACGCTAAAAGCCTAATCTCAAACCTACCCACTTCCTACAAGAGAGAAAGGGGGGGATAGTGTATAATACCCTCAACTTCGAACCAATATTCATCAGAAGTAGAGGTGTGGGTATTCTTCCACTGCAAC TGGAGGAGGCATCCAAAGGGATCGCATTGATC CCAAATCCAAGCTTTAATATTTTTCTCTCTTCTCACTCAATAATATTAATTTATTTGGGATCATGCTATCCCTTTGGATTTCTCCTTTAATGGCTTCTATAATGATGGCTCTCTCATGGATTCTGCTTGCTGCACCACAACACAAACACTTTCATATACGCCTCTAATGCT 26CACAATACAATTAAGCTCATCATACTGGTCCTGAAATTGGTGAATAAAGTTGTTTTGTGGTGGATGAGTACTGAGTAGTGGTGCCTTATTGTGGGTGGAGAGT TCCAAAGGGAT CGCATTGATCTAATTCTTGTAGATGTTTACACTTGCAAGCTTTGCATGCAATTCCTGGATTCAGATGTTATTCAGTGGTTCACTTATTGGATCATGCGATCCCTTAGGAACTTTCCATCAACTCTAAACATCTTGTTGATCCATTTGAGGAATTAATTTCATAGGTTCATATAATGGCGACTGATTCTTCTAATGGTAATGGACATCACCAAACAACAACAAAGCAACCTTCTTTGTCGTCTACACTGCGCTTATCCAAATTTTTTCAGTCCAACATGAGAATCTTGGTTACTGGAGGAGCTGGATTCATTGCGTCTTACTTAGTTGACAGATTGATGGAAAATGAAAAAAATGAGGTTATTGTCGTTGCATAGGTGCTTTCATTTTACGTTCTTCAACATTCCGAATTGAACTTCAGTGGTCCTTGCAATGGCAACGAATTCTTCTGATGTACTATCGCCGAAGCAACCTCCCTTGCCATCTCCCTTGCGTTTCTCCAAATTCTATCAGTCTAACATGAGAATCTTGATTACGGGAGGAGCTGGATTCATTGGTTCTCACCTAGTTGATAGATTGATGGAAAATGAAAAAAATGAGGTCATTGTTGCTGACAACTACTTCACTGGATCAAAGGACAACCTCAAAAAATGGATTGGTCATCCAAGATTTGAGCTTATCCGTCATGATGTCACTGAACCTTTGACGATTGAGGTTGATCAGATCTACCATCTTGCATGCCCCGCATCTCCTATTTTCTACAAATATAATCCTGTGAAGACAATAAAGACAAATGTGATTGGCACACTGAACATGCTTGGGCTTGCAAAACGAGTTGGGGCAAGGATTTTACTCACATCAACATCTGAGGTATATGGGGATCCTCTTGTGCATCCCCAACCTGAAGGCTATTGGGGCAATGTGAACCCTATTGGAGTTCGTAGTTGCTATGATGAGGGGAAACGTGTGGCTGAAACTTTGATGTTTGATTATCATAGGCAGCATGGAATAGAAATACGTGTTGCAAGAATCTTTAACACATATGGGCCGCGCATGAATATTGATGATGGACGTGTTGTCAGCAACTTCATTGCTCAAGCAATTCGTGGTGAACCCTTGACAGTCCAGTCTCCAGGAACACAAACTCGCAGTTTCTGCTATGTCTCTGATCTGGTTGATGGACTTATCCGTCTCATGGAAGGATCCGACACTGGACCAATCAACCTTGGAAATCCAGGTGAATTTACAATGCTAGAACTTGCTGAGACAGTGAAGGAGCTTATTAATCCAGATGTGGAGATAAAGGTAGTGGAGAACACTCCTGATGATCCGCGACAGAGAAAACCAATCATAACAAAAGCAATGGAATTGCTTGGCTGGGAACCAAAGGTTAAGCTGCGAGATGGGCTTCCTCTTATGGAAGAGGATTTTCGTTTGAGGCTTGGATTTGACAAAAAAAATTAACTTATTTTCGCTCCTTTTATATCTAGTCAAAATATTCAGATAATAAGTGGGATGGATTATTCTATTAAGTTTTCCTATTTTTCCTTTTCATAATTATGATACTTAGGAAGTAGGGGTGCCTGTATTTTGGCTTCCTCAATCAAGATCGTACTCTTGTTTCACAAAGCACTGCAGCAATCATGCCTTTGCAAATTTTGCCGGTAAAATTACTACTGAGTTAAAATTTTCCTATAG 28TGAAAATTACGTTTTCCCTTTTCCTTTTGTTGCCGGTTAGCACTTCAATGTAAAAATTAATTCACCATAAAGGATGGTTCGCATACAAAAGAATAAAACCTTATGAAAGGACACATGCAACGCAAAATAAAGGCATCGTTCCATAGGATATGCCGATCCTAGTGAGCCATAAATAACGTTCCCAAAGGCATTCCTCTATGTGTGTGGATCTTCCCAGTTGCAGCTGCATTACAGGGCAAGTTCTCCATTGGCAGGTAGCCACTATGATATGCATCTCATAAATATTTGCAACTTTCTTAATGTGCAATC TGCCAAAGGAGATTTGCCCAG CGATTCTCCTGCAACATCTGCTTCATGAAAACAGTATTCGTTAGTTTCTTCAATCATTCATTAGAAACATTTCTTGTACTGGTTGAAATGTTGCATCTCGAACCATTCATATGCCATATTTCCCTTGTTTTGTATTTTGGTAAAAACCATTTTTCCC 30CTGCAGAGTAAGACCTGAATTTCACTCATTGTTCCTGCCAATGTCCTTAGTTAGATAAATCTAATTTTTTCTCTCTCTAAAGTTGCATCTATAAATATGAGCCTTTCCCTTGGTGCAGATCAATTTGAGCTTTCATTACCGTTCTCATAAGCTTAGGGTGCATGCAACGGTCTCTACTTACTACTGGTTGAGAAGCTCCTTGTTGGAGAAGCAGGGCACGTGCAAGTCTCTTGGATCTCAAATGCCACTGAACCCTTTGCACGTGCTCCCCTTCTCCAACACGGGTTTCTCCCCTTGCTTTTCTCCTAACCAATTGTGTCCAGCACTTATGAGGTAATCGCTTTCCTCCTATGTCTTAATTTGGTCCTACGTAAAGATCTACAATATGCATCTTCTTTGAGATACGGGCTGAAGCATGGTACTTTTAAATTGAAGGCTTCAATAACTATATTTAGAGGGAAAATTCAACATACAAAGAAGGAAGAAGTGTTATGCATACAATATTTTACCGATGTTCTATGCGTATCAAAC ATA 32TCTATATAATTTTTTTCCTATTTTATTTTTTATTTTATTTTGTATCATATCACTTATACATCTTTTACTTTCACTCATACACTAAATTTTCGGGTGTAGGAATACTCCGGCAAAGAGAGAATAGGTTTGCTTATTTCCTAATTCTGAAGTTAGGGTACGTGCGTAATTTACTGTGTGTTCTGTGATGATGAGTTAAGTGGTCCTATTTTACATGTAACTTTTGACAATCTGTTTGGGTTGAGAATACAAATTAAGGCCCCACACCCAACTAAGCTTAGCTCTCTCCCATTTTTAGCACCCATCCCGCACCCAACTTTAAAAGCACCCTCAATTGCCTCTTCTATTATAGGAGAGTAGGCTTCAAAGCACACAAGAATATGATAAGATGAAGAAGTTCAGTGTCTCAAAATTCACCACTTCTCTTAAAACCTCCCTCATTTGTTTTTTCACACTTTCCTTTCCCTCACCACTCTCTCTATTACCTCTTGTTTGTTGTTAAGAGTACTCAGAAGAATAACTCCTCCAACCCACTTAGCATGTGGCAAAGGTGCATGCTGAGCAAGA TGGAGAAGCAGGGCACGTGCA ATTCTAACTCATGAAACCATAGAATCATCTTGTTTTTTCTTCTTTTCACTCTAACCAAATAGATTCCT CTACCTGCAG34 ACTCAAGCTTGAAGCACCAAAGTTGCAGTCGGAGGAGTCACAGATTAAATTCTTCGCTTCTTTAACCTTTGTGTTTCTCTTTTCATACCATTGTTTCTTTCCCTATAGCTGCTTTAATTTTCTTGTGAGAGTCAGAAAAGTATCACTATATCAAGTGACATGATCATCAGAATTGAATTATGTGCATGTTGTGCAAGA TGGAGAAGCAGGGCACGTGCA ATACTAACTCATGAACACTACACGGNGCGTGAACTCGGAGAATCATATTCTCTTCTGCTTCATTTCACCAACAAGAGAGATCCTATTAGTTAGTTCTTCATGTGCCCCTCTTTCCCATCATGACAACAGCACCTTATATATATTGCATTTGGAAATGTTGAACGATGAAGTTCGCTTGGCTTCTGCTCATAAATCAGCACCGAGNTTTATAGGTTATGCTCCAT

The fold-back structure of the pri-miRNA was identified in each of thesecrop plant MIR sequences using the program EINVERTED (Rice et al. (2000)Trends Genet., 16:276-277), and the results depicted in FIG. 11, whichshows the fold-back portion of the sequences, with the nucleotidepositions indicated by numbers. The fold-back portion of the MIRsequences is included in the pre-miRNA precursors processed from theseMIR genes.

The MIR sequences, the complete MIR genes which include these, and themiRNA precursors (i. e., pri-miRNAs and pre-miRNAs) processed fromthese, are useful as target sequences for gene suppression (e. g., fornuclear suppression of the production of mature miRNAs encoded by theseMIR genes) and as a source of primer or probe sequences (e. g., forprimer sequences for cloning and sequencing the promoters of these MIRgenes). The fold-back portion of the sequences has been proposed to besufficient for miRNA processing (Parizotto et al. (2004) Genes Dev.,18:2237-2242), and thus in many embodiments the region of the sequencethat contains the fold-back portion is preferably targetted forsuppression, or, alternatively, serves as the source of a sequence forsuppressing a target gene.

The mature miRNAs produced from these miRNA precursors may be engineeredfor use in suppression of a target gene, e. g., in transcriptionalsuppression by the miRNA, or to direct in-phase production of siRNAs ina trans-acting siRNA suppression mechanism (see Allen et al. (2005)Cell, 121:207-221, Vaucheret (2005) Science STKE, 2005:pe43, andYoshikawa et al. (2005) Genes Dev., 19:2164-2175, all of which areincorporated by reference herein). Plant miRNAs generally havenear-perfect complementarity to their target sequences (see, forexample, Llave et al. (2002) Science, 297:2053-2056, Rhoades et al.(2002) Cell, 110:513-520, Jones-Rhoades and Bartel (2004) Mol. Cell,14:787-799, all of which are incorporated by reference herein). Thus,the mature miRNAs can be engineered to serve as sequences useful forgene suppression of a target sequence, by replacing nucleotides of themature miRNA sequence with nucleotides of the sequence that is targettedfor suppression; see, e. g, methods disclosed by Parizotto et al. (2004)Genes Dev., 18:2237-2242 and especially U.S. Patent ApplicationPublications 2004/0053411A1, 2004/0268441A1, 2005/0144669, and2005/0037988 all of which are incorporated by reference herein. Whenengineering a novel miRNA to target a specific sequence, one strategy isto select within the target sequence a region with sequence that is assimilar as possible to the native miRNA sequence. Alternatively, thenative miRNA sequence can be replaced with a region of the targetsequence, preferably a region that meets structural and thermodynamiccriteria believed to be important for miRNA function (see, e. g., U.S.Patent Application Publication 2005/0037988). Sequences are preferablyengineered such that the number and placement of mismatches in the stemstructure of the fold-back region or pre-miRNA is preserved. Thus, anengineered miRNA or engineered miRNA precursor can be derived from anyof the mature miRNA sequences, or their corresponding miRNA precursors(including the fold-back portions of the corresponding MIR genes)disclosed herein.

An engineered miRNA precursor based on a mature miRNA (e. g., a maturemiRNA corresponding to SEQ ID NO. 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, or 35), preferably including the fold-back portion (e. g. asdepicted in FIG. 11) of the corresponding MIR sequences (e. g., SEQ IDNO. 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, or 34), is cloned and usedto evaluate engineered miRNAs in transient plant (e. g., tobacco, maize,soy, potato, and Arabidopsis) assays. Successful constructs are moved tostable transformation into a plant of interest, including maize,soybean, or potato. The sequence targetted for suppression can beendogenous or exogenous to the plant cell in which the engineered miRNAconstruct is expressed.

In a non-limiting example, engineered miRNA sequences based on thefold-back portion of SEQ ID NO. 10, 12, 14, 16, 18, 22, 24, 26, 28, 30,or 34 are engineered to target green fluorescent protein (GFP), withnucleotides of the native sequence replaced with nucleotides to match atargetted portion of the GFP sequence, while maintaining the positionand number of mismatches in the stem portion of the fold-back structure,by altering as needed the opposite strand of the stem of the fold-backstructure or pre-miRNA. The engineered miRNA sequence is placed in anexpression cassette including a suitable promoter (e. g., e35S) andterminator (e. g., Nos 3′ transcriptional terminator). As a control, asimilar gene cassette that expresses the native (non-engineeredfold-back portion of SEQ ID NO. 10, 12, 14, 16, 18, 22, 24, 26, 28, 30,or 34) is used. A third cassette is designed to express the targetsequence (GFP) and used for co-transformation with either of the miRNAcassettes. These three cassettes are inserted into binary vectors foruse in Agrobacterium-mediated transformation. Constructs are tested fortheir ability to suppress the expression of GFP in a transientco-transforrnation experiment in which leaves are transformed in plantaon wild-type maize, soybean, potato, Arabidopsis, or Nicotiana spp.plants. After four days, leaf punches corresponding to the regionsinfiltrated with Agrobacterium containing the plasmids are assayed forGFP fluorescence, which is normalized to total protein content.Constructs that express a miRNA that has been engineered to suppress theGFP gene have lower GFP expressed than the unengineered control.

Example 15

This example describes identifying novel mature miRNAs and thecorresponding MIR sequences in soy. A single small RNA was isolated andcloned using procedures based on published protocols (Llave et al.(2002) Plant Cell, 14:1605-1619, and Lau et al. (2001) Science,294:858-862). In summary, low molecular weight RNA was isolated from soy(Glycine max) leaf tissue. Adaptors were ligated followed by RT-PCR forconversion of RNA to DNA. Additional PCR amplification followed by TAcloning and sequencing led to the identification of novel mature miRNA21-mers corresponding to the DNA sequence TGAGACCAAATGAGCAGCTGA (SEQ IDNO. 36) or ATGCACTGCCTCTTCCCTGGC (SEQ ID NO. 37).

A soy cDNA contig sequence database was searched for sequencescontaining 21-mer segments identical to SEQ ID NO. 36 or SEQ ID NO. 37or to their respective complements. The sequences thus identifiedincluded SEQ ID NO. 38 (including the DNA sequence SEQ ID NO. 36corresponding to a non-conserved mature miRNA) and SEQ ID NO. 39(Gm-MIR408, including the DNA sequence SEQ ID NO. 37 corresponding tothe conserved mature miRNA miR408). The novel MIR sequences are given inTable 5, with the location of nucleotides corresponding to the maturemiRNA indicated by underlined text. TABLE 5 SEQ ID NO. Sequence 38AAAATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCATGCAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCGTCC TGAGACCAAA TGAGCAGCTGACCACATGATGCAGCTATGTTTGCTATTCAGCTGCTCATCTGTTCTCAGGTCGCCCTTGTTGGACTGTCCAACTCCTACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAAGAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAGTTACATGATTGTCTAATTGTGTTTATGGAATTGTATATTTTCAGACCAGGCACCTGTAACTAATTATAGGTACCATACCTTAAAATAAGTCCAACTAAGTCCATGTCTGTGATTTTTTAGTGTCACAAATCACAATCCATTGCCATTGGTTTTTTAATTTTTCATTGTCTGTTGTTTAACTAACTCTAGCTTTTTAGCTGCTTCAAGTACAGATTCCTCAAAGTGGAAAATGTTCTTTGAAGTCAATAAAAAGAGCTTTGATGATCATCTGCATTGTCTAAGTTGGATAAACTAATTAGAGAGAACTTTTGAACTTTGTCTACCAAATATCTGTCAGTGTCATCTGTCAGTTCTGCAAGCTGAAGTGTTGAATCCACGAGGTGCTTGTTGCAAAGTTGTGATATTAAAAGACATCTACGAAGAAGTTCAAGCAAAACTTTTTGGC 39CCGTGGTGGGCGAAGGGAATTAACGCCTATCGCGTGGCGAGAGAAGGAGCAGAACGGCAGGGGGGGGCCGGCTCCGGGGGGGCGCCCCGGTACGCACCGCGCTCTCCGAGTCCCTGGGGTCCCCCCCCCAGAACATCCTAATCGAAAAATTCAAGAGTGCATTTTGTGCGTAATGTAGTTAATTAGACAAATTTCTAATGTGAGAATCTTTCTGAGAATGAGATGTTGCTAAATATTTCGGATGTTGTCGACAAGGATGAGGTAATAATAGTTAGAGACAGGACAAAGCAGGGGAACAGGCAGAGCATGGATGGAGCTATCAACACAATATTGTCAAGAAACTGAGAGTGAGAGGAGAAATATGTTGTGGTTCTGCTC ATGCACTGCCTCTTCCCTGG CTCTGTCTCCATTTCTCCTTCCCTTATTTATTTTTTGATTTATTGAGTATGATCTGTTTTCAAATGTGTTCATAGGTTCAACTTATTAAGGTACGAACATACTCTGGGCATTGAAAACTGGTTTGACTCTTGAACATATTCCGCACCACTAATCTTTCTTGTAATCCAGGCTCACGCACGATCACTATAAGGTCCCACATTCTTAGTGGCCTAATCGTTGGAAAATGCTACTTTGGCACTACTTGATGAATTGTATGGCTGGGATTTTTTTCCCCTTGCTTGTAGAATCCTCTCAATTTATGTAACCATCGTGTACTCATTTACATGTCATCATTTTTGAATGAGATGTGATATACATAGAGCAAAAAAAAAAAAAAAATTGTATGACCTCATTTTCTGTGTTTATTTCTCTCCATCAATATCATTTTCTAAATCTCAAAATTCTCTCTTTTTTCTTAGTTGTAGAAGTTATTGTTTACTCGACTCCTCGCCTCACATCCCTCTCACCCCTCTCCCCACTACTGCCCCGCCAGCGTCACCGATGCTCTCCTTTGTGGCCGGT

The fold-back structure of the pri-miRNA was identified in these MIRsequences using the program EINVERTED (Rice et al. (2000) Trends Genet.,16:276-277), and the results shown in FIG. 12, with nucleotide positionsindicated by numbers in the fold-back portion of the sequences. Thefold-back portion of the MIR sequences is included in the pre-miRNAprecursors processed from these MIR genes.

A family of related miRNAs was cloned from the soy leaf tissue,including the abundant miRNA described above and corresponding to theDNA sequence TGAGACCAAATGAGCAGCTGA (SEQ ID NO. 36), and in lowerabundances mature miRNA 21-mers corresponding to the DNA sequenceTGAGATCAAATGAGCAGCTGA (SEQ ID NO. 40), TGAGACCAAATGAGCAGCTGT (SEQ ID NO.41), and TGAGACCAAATGACCAGCTGA (SEQ ID NO. 42), respectively, each ofwhich differs from SEQ ID NO. 36 at only one nucleotide position.

The MIR sequences, the complete MIR genes which include these, and themiRNA precursors (i. e., pri-miRNAs and pre-miRNAs) processed fromthese, the mature miRNAs transcribed from these, and miRNA recognitionsites of the mature miRNAs have various utilities as described above inExamples 12, 13, and 14 and elsewhere in this disclosure.

Example 16

This example describes identifying novel MIR sequences in maize. Publicand proprietary maize (Zea mays) genomic datasets were searched fornovel microRNA precursor sequences, starting with all pre-miRNAsequences known at the time (April 2004) using blastn and a verypermissive cutoff (e<=10,000). Hits matching a minimum length criteriawere extracted and tested (cmsearch) against all known miRNA covariancemodels (Rfam v5.1). Sequences showing significant similarity (>15 bits)to Rfam models were folded (mfold) and putative miRNAs identified. TwomicroRNA precursors in the miR166 family were thus identified, and arelisted in Table 6. These novel MIR sequences contained the consensusfold-back structure indicated by the shaded nucleotides depicted in FIG.13 (Griffiths-Jones (2004) Nucleic Acids Res., 32, Database Issue,D109-D111, which is incorporated by reference herein). TABLE 6 MIR geneSequence SEQ ID NO. 43 GTTAAGGGGTCTGTTGTCTGGTTCAAGGTCGCCACAGCAGGCAAATAAAGCCCATTTCGCGCTTAGCATG CACCATGCATGATGGGTGTACCTGTTGGTGATCTCGGACCAGGCTTCAATCCCTTTAAC SEQ ID NO. 44GTCGAGGGGAATGACGTCCGGTCCGAACGAGCCAC GGCTGCTGCTGCGCCGCCGCGGGCTTCGGACCAGGCTTCATTCCCCGTGAC

The MIR sequences, the complete MIR genes which include these, the miRNAprecursors (i. e., pri-miRNAs and pre-miRNAs) processed from these, andthe mature miRNAs transcribed from these, and miRNA recognition sites ofthe mature miRNAs have various utilities as described above in Examples12, 13, and 14 and elsewhere in this disclosure.

Example 17

This non-limiting example describes the distribution of miRNAs inspecific cells or tissues of a multicellular eukaryote (a plant).Knowledge of the spatial or temporal distribution of a given miRNA'sexpression is useful, e. g., in designing recombinant constructs to beexpressed in a spatially or temporally specific manner. This examplediscloses mature miRNA expression patterns in maize and providessequences of recognition sites for these miRNAs that are suitable forinclusion in recombinant DNA constructs useful in maize and otherplants.

Total RNA was isolated from LH244 maize plants using Trizol (Invitrogen,Carlsbad, Calif.). Seven developmental stages were used, including rootsand shoot meristems from germinating seedlings, juvenile (V1 to V2) andadult leaves (V7 to V8), stalk internode, tassel before shedding, andimmature (approximately 1″) ears. Five micrograms total RNA was resolvedon 17% PAGE-Urea as described by Allen et al. (2004) Nat. Genet.,36:1282-1290, which is incorporated by reference herein. Blots wereprobed with DNA oligonucleotides that were antisense to the small RNAsequence and end-labelled with gamma ³²P-ATP using Optikinase (USB). Theprobes used, and their respective sequences, are given in Table 7. TABLE7 SEQ ID NO. Sequence miRNA 45 GTGCTCACTCTCTTCTGTCA miR156 46TAGAGCTCCCTTCAATCCAAA miR159 47 TGGCATCCAGGGAGCCAGGCA miR160 48CTGGATGCAGAGGTTTATCGA miR162 49 TGCACGTGCCCTGCTTCTCCA miR164 50GGGGAATGAAGCCTGGTCCGA miR166 51 TAGATCATGCTGGCAGCTTCA miR167 52TTCCCGACCTGCACCAAGCGA miR168 53 TCGGCAAGTCATCCTTGGCTG miR169 54GATATTGGCGCGGCTCAATCA miR171 55 CTGCAGCATCATCAAGATTCT miR172 56GGCGCTATCCCTCCTGAGCTT miR390 57 GATCAATGCGATCCCTTTGGA miR393 58TGGGGTCCTTACAAGGTCAAGA TAS3 5′D7(+) 59 GGAGGTGGACAGAATGCCAA miR394 60GAGTTCCCCCAAACACTTCAC miR395 61 CATCAACGCTGCGCTCAATGA miR397 62CGGGGGCGACCTGAGAACACA miR398 63 AGCCAGGGAAGAGGCAGTGCA miR408

The results are shown in FIG. 14. Individual mature miRNAs wereexpressed at differing levels in specific cells or tissues. For example,Zm-miR390 was not expressed, or expressed only at low levels, in rootand adult leaf.

Example 18

This example describes recombinant DNA constructs of the invention,useful for suppressing expression of a target RNA in a specific cell ofor derived from a multicellular eukaryote such as a plant cell or ananimal cell, and methods for their use. The constructs include apromoter operably linked to DNA that transcribes to RNA including atleast one exogenous miRNA recognition site recognizable by a maturemiRNA expressed in a specific cell of a multicellular eukaryote, andtarget RNA to be suppressed in the specific cell, wherein said targetRNA is to be expressed in cells of the multicellular eukaryote otherthan the specific cell.

Strong constitutive promoters that are expressed in nearly all plantcells have been identified (e. g., CaMC 35S, OsAct), but strongspatially specific (cell- or tissue-specific) and temporally specificpromoters have been less well characterized. To limit target RNA ortransgene expression to a specific cell or tissue type in the absence ofa strong cell- or tissue-specific promoter, it may be desirable tosuppress in selected cells or tissues the expression of a transcriptunder the control of a constitutive promoter. The invention providesmethods that use recognition sequences of endogenous miRNAs to suppressexpression of a constitutively expressed target RNA in specific cells.

Methods of the invention allow spatially or temporally specificpost-transcriptional control of expression of a target RNA whereintranscription is driven by a non-specific (e. g., constitutive)promoter. The methods of the invention allow, for example, therestricted expression of a gene transcribed by a constitutive promoteror a promoter with expression beyond the desired cell or tissue type(s).Restricted expression may be spatially or temporally restricted, e. g.,restricted to specific tissues or cell types or files, or to specificdevelopmental, reproductive, growth, or seasonal stages. Where a miRNAis expressed under particular conditions (e. g., under biotic stresssuch as crowding, allelopathic interactions or pest or pathogeninfestation, or abiotic stress such as heat or cold stress, droughtstress, nutrient stress, heavy metal or salt stress), the correspondingmiRNA recognition site can be used for conditionally specificsuppression, i. e., to suppress a target RNA under the particularcondition.

For example, Zm-miR162 is poorly expressed in maize roots (see Example17 and FIG. 14), therefore, designing an expression construct to includean exogenous miRNA162 recognition site adjacent to, or within, aconstitutively expressed target RNA, may limit target RNA transcriptaccumulation in all cells of a maize plant with the exception of roots.This method has utility for all gene expression applications inmulticellular eukaryotes (plants and animals), where restrictedexpression is desired in cells wherein the given mature miRNA isexpressed.

In multicellular eukaryotes, including plants, microRNAs (miRNAs)regulate endogenous genes by a post-transcriptional cleavage mechanism,which can spatially or temporally specific. The present inventionprovides methods by which the addition of a miRNA recognition site to aconstitutively expressed transgene could be used to limit expression ofthe transgene to cells lacking, or distant to those expressing, thecomplementary mature miRNA either spatially or temporally (includingconditionally). Manipulation of these miRNA recognition sites in newtranscripts introduced into transgenic plant cells and transgenic plantsderived from these cells, is useful for altering expression patterns forthe new transgene.

In an alternative approach, an existing (native or endogenous) miRNArecognition site is mutated (e. g., by chemical mutagenesis)sufficiently to reduce or prevent cleavage (see Mallory et al. (2004)Curr. Biol., 14:1035-1046, incorporated by reference herein). In thisway a target RNA sequence with desirable effects, e. g., increased leafor seed size, can be expressed at levels higher than when the native orendogeous miRNA recognition site was present. One embodiment is toreplace a native gene with an engineered homologue, wherein a nativemiRNA has been mutated or even deleted, that is less susceptible tocleavage by a given miRNA.

One embodiment of the method is the introduction of at least oneexogenous miRNA recognition site (typically a 21 nucleotide sequence)into the 5′ or into the 3′ untranslated regions of a target RNA, orwithin the target RNA. Where the target RNA includes coding sequence,the at least one exogenous miRNA recognition site can be introduced intothe coding region of the target RNA. This results in the reducedexpression of the target RNA in tissues or cell types that express thecorresponding mature miRNA. By including a recognition sitecorresponding to a mature miRNA in a target RNA transcript, it ispossible to modulate the target RNA's expression in such a way that evenunder the control of a constitutive promoter, the target RNA isexpressed only in selected cells or tissues or during selected temporalperiods. This allows both the high levels of expression obtainable withstrong constitutive promoters, and spatial or temporal limiting of suchexpression.

Any miRNA recognition site may be used, preferably where the expressionof the corresponding mature miRNA has been determined to suit thedesired expression or suppression of the target RNA. Numerous miRNArecognition sequences are known. See, for example, Jones-Rhoades andBartel (2004). Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell,110:513-520, Allen et al. (2004) Nat. Genet., 36:1282-1290, which areincorporated by reference herein). Also see the ASRP database online(Gustafson et al. (2005) Nucleic Acids Res., 33:D6379-D640).Non-limiting examples of miRNA recognition sites useful in constructsand methods of the invention include those provided in Table 8, whichgives the recognition site sequences for the indicated miRNA family andindicates the distribution among “all plants” (i. e., lower plants,monocots, and dicots), monocots and/or dicots. The plant species fromwhich the miRNA was identified and the abbreviations used were:Arabidopsis thaliana (At), Glycine max (Gm), Gossypium hirsutum (Gh),Hordeum vulgare (Hv), Lycopersicum esculentum (Le), Lotus corniculatusvar. japonicus (synonymous with “Lotus japonicus”) (Lj), Medicagotruncatula (Mt), Mesembryanthemum crystallinum (Mc), Oryza sativa (Os),Pennisetum glaucum (Pg), Phaseolus vulgaris (Pv), Populus tremula (Pt),Saccharum officinarum (So), Sorghum bicolor (Sb), Theobroma cacao (Tc),Triticum aestivum (Ta), Vitis vinifera (Vv), and Zea mays (Zm). TABLE 8SEQ ID miRNA Recognition Recognition Site NO. Site Sequence miR156family recognition sequence - all plants 64 At1g27370GUGCUCUCUCUCUUCUGUCA 65 At1g53160 CUGCUCUCUCUCUUCUGUCA 66 At2g33810UUGCUUACUCUCUUCUGUCA 67 At3g15270 CCGCUCUCUCUCUUCUGUCA miR159 familyrecognition sequence - all plants 68 At5g06100 UGGAGCUCCCUUCAUUCCAAU 69At2g26960 UCGAGUUCCCUUCAUUCCAAU 70 At4g26930 AUGAGCUCUCUUCAAACCAAA 71At2g26950 UGGAGCUCCCUUCAUUCCAAG 72 At2g32460 UAGAGCUUCCUUCAAACCAAA 73At3g60460 UGGAGCUCCAUUCGAUCCAAA 74 At5g55020 AGCAGCUCCCUUCAAACCAAA 75PvMYB CAGAGCUCCCUUCACUCCAAU 76 VvMYB UGGAGCUCCCUUCACUCCAAU 77 HvMYB33UGGAGCUCCCUUCACUCCAAG 78 OsMYB33 UGGAGCUCCCUUUAAUCCAAU miR160 familytarget sequences - all plants 79 At1g77850 UGGCAUGCAGGGAGCCAGGCA 80At2g28350 AGGAAUACAGGGAGCCAGGCA 81 At4g30080 GGGUUUACAGGGAGCCAGGCA 82OsARF AGGCAUACAGGGAGCCAGGCA 83 LjARF AAGCAUACAGGGAGCCAGGCA miR161 familytarget sequences - Arabidopsis 84 At5g41170 ACCUGAUGUAAUCACUUUCAA 85At1g06580 CCCGGAUGUAAUCACUUUCAG 86 At1g63150 UUGUUACUUUCAAUGCAUUGA 87At5g16640 CCCUGAUGUAUUUACUUUCAA 88 At1g62590 UAGUCACGUUCAAUGCAUUGA 89At1g62670 CCCUGAUGUAUUCACUUUCAG 90 At1g62860 CCCUGAUGUUGUUACUUUCAG 91At1g62910 UAGUCACUUUCAGCGCAUUGA 92 At1g62930 UCCAAAUGUAGUCACUUUCAG 93At1g63080 UCCAAAUGUAGUCACUUUCAA 94 At1g63130 UCCAAAUGUAGUCACUUUCAG 95At1g63400 UCCAAAUGUAGUCACUUUCAA 96 At1g63230 UUGUAACUUUCAGUGCAUUGA 97At1g63330 UAGUCACGUUCAAUGCAUUGA 98 At1g63630 UUGUUACUUUCAGUGCAUUGA 99At1g64580 CCCUGAUGUUGUCACUUUCAC 100 At2g41720 UUGUUACUUACAAUGCAUUGA 101At1g63070 UAGUCUUUUUCAACGCAUUGA miR162 family target sequences -monocots and dicots 102 At1g01040 CUGGAUGCAGAGGUAUUAUCGA 103 PtDCL1CUGGAUGCAGAGGUCUUAUCGA 104 OsDCL1 CUGGAUGCAGAGGUUUUAUCGA miR163 familytarget sequences - Arabidopsis 105 At1g66700 AUCGAGUUCCAAGUCCUCUUCAA 106At1g66720 AUCGAGUUCCAGGUCCUCUUCAA 107 At3g44860 AUCGAGUUCCAAGUUUUCUUCAAmiR164 family target sequences - monocots and dicots 108 At1g56010AGCACGUACCCUGCUUCUCCA 109 At5g07680 UUUACGUGCCCUGCUUCUCCA 110 At5g53950AGCACGUGUCCUGUUUCUCCA 111 At5g61430 UCUACGUGCCCUGCUUCUCCA 112 At5g39610CUCACGUGACCUGCUUCUCCG 113 OsNAC1 CGCACGUGACCUGCUUCUCCA 114 MtNACCUUACGUGUCCUGCUUCUCCA 115 GmNAC CUUACGUGCCCUGCUUCUCCA 116 LeNACGCCACGUGCACUGCUUCUCCA miR165/166 family target sequences - all plants117 At1g30490 UUGGGAUGAAGCCUGGUCCGG 118 At5g60690 CUGGGAUGAAGCCUGGUCCGG119 At1g52150 CUGGAAUGAAGCCUGGUCCGG 120 PtHDZIPIII CCGGGAUGAAGCCUGGUCCGGmiR167 family target sequences - monocots and dicots 121 At1g30330GAGAUCAGGCUGGCAGCUUGU 122 At5g37020 UAGAUCAGGCUGGCAGCUUGU 123 OsARF6AAGAUCAGGCUGGCAGCUUGU miR168 family target sequences - all plants 124At1g48410 UUCCCGAGCUGCAUCAAGCUA miR169 family target sequences - allplants 125 At1g17590 AAGGGAAGUCAUCCUUGGCUG 126 At1g54160ACGGGAAGUCAUCCUUGGCUA 127 At1g72830 AGGGGAAGUCAUCCUUGGCUA 128 At3g05690AGGCAAAUCAUCUUUGGCUCA 129 At3g20910 GCGGCAAUUCAUUCUUGGCUU 130 At5g12840CCGGCAAAUCAUUCUUGGCUU 131 At3g14020 AAGGGAAGUCAUCCUUGGCUA 132 ZmHAP2GUGGCAACUCAUCCUUGGCUC 133 VvHAP2 UGGGCAAUUCAUCCUUGGCUU 134 OsHAP2AUGGCAAAUCAUCCUUGGCUU 135 GmHAP2 UAGGGAAGUCAUCCUUGGCUC 136 GhHAP2CUGGGAAGUCAUCCUUGGCUC miR170/171 family target sequences - all plants137 At2g45160 GAUAUUGGCGCGGCUCAAUCA miR172 family target sequences - allplants 138 At4g36920 CUGCAGCAUCAUCAGGAUUCU 139 At2g28550CAGCAGCAUCAUCAGGAUUCU 140 At5g60120 AUGCAGCAUCAUCAGGAUUCU 141 At5g67180UGGCAGCAUCAUCAGGAUUCU 142 At2g39250 UUGUAGCAUCAUCAGGAUUCC 143 At3g54990UUGCAGCAUCAUCAGGAUUCC miR319 family target sequences - all plants 144At4g18390 CAGGGGGACCCUUCAGUCCAA 145 At1g53230 GAGGGGUCCCCUUCAGUCCAU 146At3g15030 GAGGGGUCCCCUUCAGUCCAG 147 At2g31070 AAGGGGUACCCUUCAGUCCAG 148At1g30210 UAGGGGGACCCUUCAGUCCAA 149 OsPCF5 GAGGGGACCCCUUCAGUCCAG 150OsPCF8 UCGGGGCACACUUCAGUCCAA miR393 family target sequences - monocotsand dicots 151 At1g12820 AAACAAUGCGAUCCCUUUGGA 152 At4g03190AGACCAUGCGAUCCCUUUGGA 153 At3g23690 GGUCAGAGCGAUCCCUUUGGC 154 At3g62980AGACAAUGCGAUCCCUUUGGA miR394 family target sequences - monocots anddicots 155 At1g27340 GGAGGUUGACAGAAUGCCAAA miR395 family targetsequences - monocots and dicots 156 At5g43780 GAGUUCCUCCAAACACUUCAU 157At3g22890 GAGUUCCUCCAAACUCUUCAU 158 At5g10180 AAGUUCUCCCAAACACUUCAAmiR396 family target sequences - monocots and dicots 159 At2g22840UCGUUCAAGAAAGCCUGUGGAA 160 At2g36400 CCGUUCAAGAAAGCCUGUGGAA 161At4g24150 UCGUUCAAGAAAGCAUGUGGAA 162 At2g45480 ACGUUCAAGAAAGCUUGUGGAA163 At3g52910 CCGUUCAAGAAAGCCUGUGGAA miR397 family target sequences -monocots and dicots 164 At2g29130 AAUCAAUGCUGCACUCAAUGA 165 At2g38080AGUCAACGCUGCACUUAAUGA 166 At2g60020 AAUCAAUGCUGCACUUAAUGA miR398 familytarget sequences - monocots and dicots 167 At1g08830AAGGGGUUUCCUGAGAUCACA 168 At2g28190 UGCGGGUGACCUGGGAAACAUA 169 At3g15640AAGGUGUGACCUGAGAAUCACA miR173 family target sequences - Arabidopsis 170AtTAS1a GUGAUUUUUCUCAACAAGCGAA 171 AtTAS1c GUGAUUUUUCUCUACAAGCGAA 172AtTAS2 GUGAUUUUUCUCUCCAAGCGAA miR399 family target sequences - monocotsand dicots 173 At2g33770 UAGGGCAUAUCUCCUUUGGCA 174 At2g33770UUGGGCAAAUCUCCUUUGGCA 175 At2g33770 UCGAGCAAAUCUCCUUUGGCA 176 At2g33770UAGAGCAAAUCUCCUUUGGCA 177 At2g33770 UAGGGCAAAUCUUCUUUGGCA 178 OsE2UBCUAGGGCAAAUCUCCUUUGGCA 179 OsE2UBC CUGGGCAAAUCUCCUUUGGCA 180 OsE2UBCUCGGGCAAAUCUCCUUUGGCA 181 OsE2UBC CCGGGCAAAUCUCCUUUGGCA 182 PtE2UBCGCGGGCAAAUCUUCUUUGGCA 183 MtE2UBC AAGGGCAAAUCUCCUUUGGCA 184 TaE2UBCUAGGGCAAAUCUCCUUUGGCG 185 TaE2UBC CUGGGCAAAUCUCCUUUGGCG 186 TaE2UBCUUCGGCAAAUCUCCUUUGGCA miR403 family target sequences - dicots 187At1g31280 GGAGUUUGUGCGUGAAUCUAAU miR390 family target sequences - allplants 188 At3g17185 CUUGUCUAUCCCUCCUGAGCUA 189 SbTAS3UAUGUCUAUCCCUUCUGAGCUG 190 SoTAS3 UAUGUCUAUCCCUUCUGAGCUA 191 ZmTAS3UAUGUCUAUCCCUUCUGAGCUG 192 OsTAS3 UCGGUCUAUCCCUCCUGAGCUG 193 PgTAS3UUAGUCUAUCCCUCCUGAGCUA 194 VvTAS3 AUUGCCUAUCCCUCCUGAGCUG 195 TcTAS3CCUUGCUAUCCCUCCUGAGCUG 196 LeASR CUUGUCUAUCCCUCCUGAGCUG 197 ZmTAS3CCCUUCUAUCCCUCCUGAGCUA 198 PtTAS3 CUUGUCUAUCCCUCCUGAGCUA 199 OsTAS3CCCUUCUAUCCCUCCUGAGCUA 200 TaTAS3 CCCUUCUAUCCCUCCUGAGCUA 201 HvTAS3CCUUUCUAUCCCUCCUGAGCUA 202 PtTAS3 CCUGUCUAUCCCUCCUGAGCUA 203 McTAS3UGUGUCUAUCCCUCCUGAGCUA miR447 family target sequences - Arabidopsis 204At5g60760 UGACAAACAUCUCGUCCCCAA 205 At3g45090 UGACAAACAUCUCGUUCCUAAmiR408 family target sequences - monocots and dicots 206 At2g02850CCAAGGGAAGAGGCAGUGCAU 207 At2g30210 ACCAGUGAAGAGGCUGUGCAG 208 At2g47020GCCAGGGAAGAGGCAGUGCAU 209 At5g05390 GCCGGUGAAGAGGCUGUGCAA 210 At5g07130GCCGGUGAAGAGGCUGUGCAG TAS3 ta-siRNA target sequences - monocots anddicots 211 At2g33860a AGGGUCUUGCAAGGUCAAGAA 212 At5g60450aAAGGUCUUGCAAGGUCAAGAA 213 OsARF3-like GAGGUCUUGCAAGGUCAAGAA 214OsARF2-like ACGGUCUUGCAAGGUCAAGAA TAS1/TAS2 target sequences -Arabidopsis thaliana 215 Atg12770 AGAACUAGAGAAAGCAUUGGA 216 Atg12770AGAGUAAGAUGGAGCUUGAUA 217 Atg163130 AGAUGGUGGAAAUGGGAUAUC 218 At1g63230UUGUUGAUCGUAUGGUAGAAG 219 At1g62930 GGUAUUCGAGUAUCUGCAAAA

Thus, a transgenic plant expressing a recombinant DNA construct that,under the control of a constitutive promoter (e. g., a 35S promoter)transcribes to RNA containing a Zm-miR390 recognition site and a targetRNA would be expected to show suppression of the target RNA expressionin root and adult leaf, relative to expression in other tissues.

In another example, Zm-miR172 was expressed at high levels in stalk, andnot expressed, or expressed only at low levels, in other tissues. Atransgenic plant expressing a construct that, under the control of astrong constitutive promoter (e. g., a CaMV 35S promoter) transcribes toRNA containing a Zm-miR172 recognition site and a target RNA would beexpected to express that target RNA at higher levels in tissues otherthan stalk (where expression of the target RNA would be suppressed).

To illustrate use of the constructs and methods of the invention tocontrol expression of a gene of interest, a reporter gene is used as thegene of interest itself, or as a surrogate for the gene of interest. Forexample, where expression of a reporter gene (e. g., green fluorescentprotein, GFP) is desired in maize stalk and immature ear tissue, amiR156 target site is included in a GFP expression cassette andexpressed in a stably transgenic maize plant under the control of theCaMV 35S promoter. In other tissues (e. g., roots, leaves, and tassel),GFP expression is suppressed. The suppression phenotype may be limitedto very specific cell types within the suppressed tissues, withneighboring cells showing expression or a gradient of expression of GFPadjacent to those cells expressing the mature miR156.

In another example, a strong constitutive promoter is used to driveexpression of a Bacillus thuringiensis insecticidal protein or proteinfragment (“Bt”), where a recognition site for a miRNA expressed inpollen is included in the construct, resulting in strong expression intissues of the plant except for the pollen.

One specific, non-limiting example of the method is the inclusion of therecognition site for a miRNA that is not expressed in roots to arecombinant DNA construct including a target RNA of which expression isdesired only in the roots. A strong constitutive promoter (e. g.,enhanced 35S) can still be used, but the target RNA's expression is nowrestricted to the cells that that do not express the correspondingmature miRNA. A specific example of this approach is the inclusion of amaize miRNA162, maize miRNA164, or maize miRNA390 recognition site in arecombinant DNA construct for the expression of a Bacillus thuringiensisinsecticidal protein or protein fragment (“Bt”, see, for example, theBacillus thuringiensis insecticidal sequences and methods of use thereofdisclosed in U.S. Pat. No. 6,953,835 and in U.S. Provisional PatentApplication No. 60/713,111, filed on 31 Aug. 2005, which areincorporated by reference herein) as the target RNA, e. g., in aconstruct including the expression cassette e35S/Bt/hsp17. These miRNAs(e.g., miRNA162, miRNA164, or miRNA390) are not substantially expressedin maize roots but are expressed in most other tissues. Including one ormore of these recognition sites within the expression cassette reducesthe expression of transcripts in most tissues other than root, butmaintains high Bt target RNA expression levels in roots, such as isdesirable for control of pests such as corn rootworm. In one embodiment,combinations of different miRNA recognition sites are included in theconstruct in order to achieve the desired expression pattern.

Non-limiting specific examples of transcribable DNA sequence includingan exogenous miRNA recognition site are depicted in FIG. 15 and FIG. 16.FIG. 15 depicts chloroplast-targeted TIC809 with a miRNA 162 recognitionsite (in bold text) located in the 3′ untranslated region (SEQ ID NO.220). FIG. 16 depicts non-targeted TIC809 with a miRNA164 recognitionsite (in bold text) located in the 3′ untranslated region (SEQ ID NO.221).

Example 19

This example describes a crop plant miRNA gene with tissue-specificexpression, and identification of the miR gene promoter. Moreparticularly, this example describes identification of a maize miR 167promoter sequence with endosperm-specific expression. A member of themiR167 family (SEQ ID NO. 4) was found to represent about a quarter ofthe small RNA population cloned from developing maize endosperm asdescribed in Example 13. To determine whether a single miR167 genefamily member is responsible for the observed strong endospermexpression, several miR167 genes were analyzed by RT-PCR. Nine Zea maysmiR167 stem-loop sequences were found in the public miRNA registry(‘miRBase”, available on line at microrna.sanger.ac.uk/sequences),listed as miR167a through miR167i. Tissue-specific RT-PCR was performedfor several of the Z. mays miR167 sequences using gene-specific primersfor first strand cDNA synthesis followed by PCR with gene-specificprimer pairs. Expression of miR167g was strong and tissue-specific forendosperm (15, 20 days after pollination).

To determine whether miR167g is abundantly expressed in endosperm,Northern blots of maize (LH59) were prepared. The blot was probed withan end-labeled mature miR167 22-mer LNA probe (FIG. 17A), stripped, andre-probed with a ˜400 bp miR167g gene-specific probe (FIG. 17B). Thestrong endosperm signal observed indicated that miR167g is largelyresponsible for endosperm-enhanced expression. Transcription profilingof maize tissues corroborated the Northern blot results (FIG. 17C); thetranscript corresponding to miR167g was abundantly and specificallyexpressed in endosperm tissue.

A GenBank publicly available 804 base pair cDNA sequence (annotated as“ZM_BFb0071120.r ZM_BFb Zea mays cDNA 5′, mRNA sequence”) and having theaccession number DR827873.1 (GI:71446823) is incorporated here byreference. This sequence includes a segment corresponding to the maturemiR167g (SEQ ID NO. 4). Using the public sequence, bioinformaticanalysis was performed on proprietary maize genomic sequence. A 4.75kilobase genomic cluster including sequence from maize inbred line B73was identified as containing predicted gene sequences for miR167a andmiR167g. A 486 base pair region between the two miR167 genes wasidentified as having homology to an expressed sequence tag (EST)sequence. Promoter motifs were identified in the upstream sequences ofboth (miR167a and miR167g) predicted transcripts. A region of 1682 basepairs (SEQ ID NO. 222) between the predicted miR167a and miR167gtranscripts, and a smaller region of 674 base pairs (SEQ ID NO. 223)between the EST and the predicted miR167g transcript was identified asmiR167g promoter sequences. Subsets of these sequences (e. g., at leastabout 50, about 100, about 150, about 200, about 250, or about 300nucleotides of SEQ ID NO. 222 or SEQ ID NO. 223, or fragments of atleast about 50, about 100, about 150, and about 200 contiguousnucleotides having at least 85%, at least 90%, or at least 95% identityto a segment of SEQ ID NO. 222 or SEQ ID NO. 223) are also useful aspromoters; their promoter effects are demonstrable by procedures wellknown in the art (e. g., to drive expression of a reporter gene such asluciferase or green fluorescent protein). The annotation map, includinglocations of the miR167a and miR167g genes and mature miRNAs, andpromoter elements (e. g., TATA boxes), of this genomic cluster is shownin FIG. 18. The annotation map also shows the location ofauxin-responsive factor (ARF) motifs or auxin response elements with thesequence TGTCTC (SEQ ID NO. 224), which indicates that auxin mayregulate expression of miR167g. Mature miR167 miRNAs are complementaryto ARF6 and ARF8 (which encode activating ARFs) and have been proposedto regulate auxin homeostasis; see, for example, Rhoades et al. (2002)Cell, 110:513-520, Bartel and Bartel (2003) Plant Physiol., 132:709-717,Ulmasov et al. (1999) Proc. Natl. Acad. Sci. USA, 96:5844-5849, andMallory et al. (2005) Plant Cell, 17:1360-1375, all of which areincorporated by reference herein.

In addition to the miR167g promoter sequences (SEQ ID NO. 222 and SEQ IDNO. 223) identified from maize inbred line B73, two additional miR167gpromoter sequences (SEQ ID NO. 225 and SEQ ID NO. 226) were amplifiedfrom the maize inbred line LH244. The 3′ ends of SEQ ID NO. 225 and SEQID NO. 226 were determined experimentally by 5′ RACE (rapidamplification of cDNA ends, Invitrogen Corporation, Carlsbad, Calif.) ofmiR167g. The 5′ end of the 768 base pairs sequence (SEQ ID NO. 225)corresponds to the end of a GenBank publicly available 481 base paircDNA sequence (annotated as “QCG17c03.yg QCG Zea mays cDNA cloneQCG17c03, mRNA sequence”) and having the accession number CF035345.1(GI:32930533). The 5′ end of the 407 base pairs sequence (SEQ ID NO.226) corresponds to the end of a GenBank publicly available 746 basepair cDNA sequence (annotated as “MEST991_A06.T7-1 UGA-ZmSAM-XZ2 Zeamays cDNA, mRNA sequence”) and having the accession number DN214085.1(GI:60347112).

The miR167g promoter sequences, miR167g gene, mature miR167g microRNA,and miR167g recognition site described herein have various utilities asdescribed in Examples 12, 13, 14, and 18, and elsewhere in thisdisclosure. In particular, a miR167g promoter is useful as anendosperm-specific promoter, and can be used, for example to replace themaize B32 promoter used in the recombinant DNA construct described inExample 4 (also see FIG. 5B). In another utility, the miR167g sequenceor mature miR167g (or a precursor thereof) is engineered to suppress atarget gene, especially where suppression is to be endosperm-specific.The miR167g recognition site is useful, e. g., in constructs for geneexpression where the gene is to be expressed in tissues other thanendosperm.

Example 20

This example describes a recombinant DNA construct including atranscribable engineered miRNA precursor designed to suppress a targetsequence, wherein the transcribable engineered miRNA precursor isderived from the fold-back structure of a MIR gene, preferably a maizeor soybean MIR sequence.

MicroRNA genes were cloned essentially as described in Example 15 frommaize. These included a ZmMIR159a sequence (SEQ ID NO. 227) and aZmMIR164e (SEQ ID NO. 228); the sequences are provided in Table 9, withthe location of nucleotides corresponding to the mature miRNA indicatedby underlined text. TABLE 9 Zea mays MIR sequence MIR159aGCATCTGCTGTTCTTTATTTCTATACATACATATATACTATCAC (SEQ ID NO. 227)CGGTTATTGCTTCTCTATTCTGTCCGAGTACTTTACGGTGTTCCGCACATAGATCTCGTGGCCGGCGGTTTTGCGCTTTCGCTTGCGTTTCTTGGCCCTGCTGGTGTTTGACCGGACCGAACGGGGGCAGATCGATGCTTTGGGTTTGAAGCGGAGCTCCTATCATTCCAATGAAGGGTCGTTCCGAAGGGCTGGTTCCGCTGCTCGTTCATGGTTCCCACTATCCTATCTCATCATGTGTATATATGTATTCCATGGGGGAGGGTTTCTCTCGTCTTTGAGATAGGCTTGTGGTTTGCATGACCGAGGAGCTGCACCGCCCCCTTGCTGGCCGCTCTTTGGATTGAAGGGAGCTCTGCATCCTGATCCACCCCTCCATTTTTTTTTGCTTGTTGTGTCCTTCCTGGGACCTGAGATCTGAGGCTCGTGGTGGCTCACTG MIR164eCCTTGTATGTTCTCCGCTCACTCCCCCATTCCACTCTCATCCATCTCTC (SEQ ID NO. 228)AAGCTACACACATATAAAAAAAAAAGAGTAGAGAAGGACCGCCGTTAGAGCACTTGATGCATGCGTACGTCGATCCGGCGGACCGATCTGCTTTTGCTTGTGTGCTTGGTGAGAAGGTCCCTGTTGGAGAAGCAGGGCACGTGCAGAGACACGCCGGAGCACGGCCGCCGCCGATCTACCGACCTCCCACACCTGCCTTGTGGTGTGGGGGTGGAGGTCGTCGGTGGAAGCGATAGCTGTCGTTGTTGCTTCGATGTTGTTAGCTCCTCCTGCACGTGCTCCCCTTCTCCACCACGGCCTTCTCACCACCCTCCTCCCCCGGCGGCGGCGGCGGCGGACCGCCCTTGCCGCGATCAATAATGAAACCAAAAGCCGACAGTATTTGAGCAGGAAATACAAGAGGCGGATATCCCACTGCTAGCACTTCTGCGTTGATCATGtTCATCTGGAACAAAATAATACTCGGCGACTTTACAGCGAGTGCAGCATG

An engineered miRNA, “MIR159a-CPB.miR1”, based on cloned SEQ ID NO. 227,was designed to target a vacuolar ATPase sequence from Colorado potatobeetle and had the sequence

GCATCTGCTGTTCTTTATTTCTATACATACATATATACTATCACCGGTTATTTGCTTCTCTATTCTGTCCGAGTACTTTACGGTGTTCCGCACATAGATCTCGTGGCCGGCGGTTTTGCGCTTTCGCTTGCGTTTCTTGGCCCTGCTGGTGTTTGACCGGACCGAACGGGGGCAGATCGATGCTTTGGGTTTGAAGatacGtggCaAaacTaggAATGAAGGGTCGTTCCGAAGGGCTGGTTCCGCTGCTCGTTCATGGTTCCCACTATCCTATCTCATCATGTGTATATATGTATTCCATGGGGGAGGGTTTCTCTCGTCTTTGAGATAGGCTTGTGGTTTGCATGACCGAGGAGCTGCACCGCCCCCTTGCTGGCCGCTCTTTCCTGGTTCTGCCACGTATCATCCTGATCCACCCCTCCATTTTTTTTTGCTTGTTGTGTCCTTCCTGGGACCTGAGATCTGAGGCTCGTGGTGGCTCACTG (SEQ ID NO.229, where the nucleotides corresponding to the engineered mature miRNAare indicated by bold underlined text, and the nucleotides included inthe complementary strand of the miRNA hairpin are indicated bylower-case text). A recombinant DNA construct containing this engineeredmiRNA (SEQ ID NO. 229), was made and expressed in tobacco (N.benthamiana) using a transient in planta expression assay as in Llave etal. (2002) Plant Cell, 14:1605-1619 and Palatnik et al. (2003) Nature,425:257-263, which are incorporated by reference herein. Briefly,Agrobacterium tumefaciens containing a binary expression vector wasgrown to late log phase, VIR genes induced, and all desired combinationsof expression vectors mixed to a final optical density (600 nanometers)of 0.5. A GFP expression vector was used to equalize all mixes to thesame optical density. Agrobacterium mixes were infiltrated into N.benthamiana using a syringe applied with slight pressure to the bottomsurface of two to three leaves per plant leaf. Inoculated leaves wereharvested 48 hours after infiltration. All assays were performed intriplicate, with a single plant per replicate. The predicted matureengineered miRNA processed from the precursor sequence SEQ ID NO. 229has the sequence UUUCCUGGUUCUGCCACGUAU (SEQ ID NO. 230), which has aReynolds score of 4 (where values range from −1 to 10 and a higher scoreis predictive of efficacy; see Reynolds et al. (2004) NatureBiotechnol., 22:326-330, which is incorporated by reference in itsentirety herein), a functional asymmetry score of −1.1 (where a negativevalue predicts incorporation into the RISC complex, see Khvorova et al.(2003) Cell, 115:209-216, which is incorporated by reference herein),and was observed to be efficiently processed (FIG. 19B).

This approach is useful with other plant mature miRNA and miRNAprecursor sequences, which can be engineered to silence various targetgenes of the plant or of a pest or pathogen of the plant. Thus, anotherengineered miRNA, “MIR159a-CRW.miR1”, also based on cloned SEQ ID NO.227, is designed to target a vacuolar ATPase sequence from corn rootwormand had the sequence

GCATCTGCTGTTCTTTATTTCTATACATACATATATACTATCACCGGTTATTGCTTCTCTATTCTGTCCGAGTACTTTACGGTGTTCCGCACATAGATCTCGTGGCCGGCGGTTTTGCGCTTTCGCTTGCGTTTCTTGGCCCTGCTGGTGTTTGACCGGACCGAACGGGGGCAGATCGATGCTTTGGGT-TTGAAGTCTCTGGCAGTAACTGACAATGAAGGGTCGTTCCGAAGGGCTGGTTCCGCTGCTCGTTCATGGTTCCCACTATCCTATCTCATCATGTGTATATATGTATTCCATGGGGGAGGGTTTCTCTCGTCTTTGAGATAGGCTTGTGGCTTTGCATGACCGAGGAGCTGCACCGCCCCCTTGCTGGCCGCTCTTTGTCCGTTTCTGCCAGAGACATCCTGATCCACCCCTCCATTTTTTTTTGCTTGTTGTGTCCTTCCTGGGACCTGAGATCTGAGGCTCGTGGTGGCTCACTG (SEQ IDNO. 231, where the nucleotides corresponding to the engineered maturemiRNA are indicated by bold underlined text). The Western corn rootworm(Diabrotica virgifera) vacuolar ATPase sequence selected for suppressionhas the sequence

AGAAGCCTGGCAATTTCCAAGGTGATTTTGTCCGTTTCTGCCAGAGATGCTTTACCTACCAGCTGCACAATTTCGGCTAGATCATCTTCTTCCTGAAGAATTTCCTTAACTTT-GGTTCTAAGAGGAATAAACTCTTGGAAGTTTTTGTCATAAAAGTCGTCCAATGCTCTTAAATATTTGGAATATGATCCAAGCCAGTCTACTGAAGGGAAGTGCTTACGTTGGGCAAG (SEQ ID NO. 232). The predicted mature engineeredmiRNA processed from the precursor sequence SEQ ID NO. 229 has thesequence UUUGUCCGUUUCUGCCAGAGA (SEQ ID NO. 233), which has a Reynoldsscore of 6 and a functional asymmetry score of −3.2. This genesuppression element is tested in Agrobacterium-mediated transient assaysin tobacco for expression of the engineered miRNA, and then stablytransformed into maize to test for efficacy in controlling cornrootworm. These engineered miRNAs or miRNA precursors can be included invarious recombinant DNA constructs of the invention, e. g., in aconstruct including the engineered miRNA or miRNA precursor embeddedwithin an intron flanked on one or on both sides by non-protein-codingDNA, or in combination with a miRNA recognition site, or with anaptamer.

Example 21

Current criteria for miRNA identification have emphasized phylogeneticconservation of miRNAs across species, and thus few non-conserved orspecies-specific miRNAs in plants have been characterized in plants.This example describes identifying five novel non-conserved miRNAs andthe corresponding MIR sequences from a size-fractionized cDNA libraryconstructed from soybean leaves. Criteria for miRNA identificationincluded: (1) a cloned 21-nt small RNA, and possible miRNA* (strandcorresponding to the miRNA) at a lower abundance, (2) containment of themiRNA/miRNA* duplex wholly within a short, imperfect foldback structure,(3) derivation of the miRNA from an RNA Pol II non-protein-codingtranscript, and (4) presence of a complementary target site in a codinggene; see Ambros et al. (2003) RNA, 9: 277-279, which is incorporated byreference herein.

Small RNAs were extracted from adaptor-containing raw sequences andtheir strands were determined. This sequence set was filtered to removesmall RNA sequences that were virus, tRNA, rRNA, chloroplast andmitochondria RNAs, and transgene, resulting in a filtered set of 381,633putative miRNA sequences. Small RNAs not originating from the abovesources and not homologous to known miRNAs were mapped to referencesoybean cDNA sequences. For the mapped cDNA sequences with lowprotein-coding content, a cDNA sequence fragment of about 250nucleotides, containing the putative miRNA, was folded using RNA Folder.The foldback structure was examined to check if the small RNA waslocated in the stem, and if an extensively (but not perfectly)complementary small RNA with lower abundance was located in the oppositeside of the stem. The potential targets of the small RNA are predictedbased on rules modified from Jones-Rhoades and Bartel (2004) Mol. Cell,14:787-799, and Zhang (2005) Nucleic Acids Res., 33:W701-704, which areincorporated by reference herein. Table 10 lists the five novelnon-conserved miRNAs cloned from soy leaf tissue, and for each thecorresponding miRNA* and precursor pri-miRNA(s); abundance (“abund”) isgiven as the number of times the sequence occurred in a total of 381,633sequences. TABLE 10 miRNA miRNA miRNA* precursor SEQ SEQ SEQ ID ID IDNO. sequence abund NO. sequence abund NO. 234 UGAGACCAAAUGAGCAGCUGA94123 235 GCUGCUCAUCUGUUCUCAGG 26 236 237 UAGAAGCUCCCCAUGUUCUCA 7259 238GAGCAUGGGUAACUUCUAU 24 239 240 UGUUGCGGGUAUCUUUGCCUC 4127 241GGCGUAGAUCCCCACAACAG 9 242 243 UGCGAGUGUCUUCGCCUCUGA 3778 244GGAGGCGUAGAUACUCACACC 70 245 246 UUGCCGAUUCCACCCAUUCCUA 3733 247GCUGCUCAUCUGUUCUCAGG 93 248, 249

For each novel soy miRNA, the fold-back structure of the miRNA precursorsequence(s) was predicted by an algorithm (“RNAFolder”, based onRNAfold, publicly available atwww.tbi.univie.ac.at/˜ivo/RNA/RNAfold.html), and the miRNA precursortranscription profile obtained when available, as listed in Table 11.Examples of predicted targets (recognition sites) in soybean and theirexpression pattern identified were identified for two of the miRNAs (SEQID NO. 234 and SEQ ID NO. 237). TABLE 11 miRNA predicted predictedprecursor G. max target target miRNA miRNA miRNA precursor transcription(recognition site) expression SEQ ID NO. precursor fold-back profilesequence pattern 234 236 see FIG. 20A see FIG. 20B polyphenol oxidasesee FIG. (SEQ ID NO. 250) 20C 237 239 see FIG. 21A — polyphenol oxidasesee FIG. (SEQ ID NO. 251) 21B 240 242 see FIG. 22 — — — 243 245 see FIG.23 — — — 246 248, 249 see FIG. 24A see FIG. 24B — —

In addition, target (recognition site) sequences for each novel soymiRNA were from in-house (“MRTC”) soy databases, as listed in Table 12.TABLE 12 Glycine max target Location of (recognition target target site)SEQ ID MRTC (recognition (recognition site) NO. designation site)sequence score mismatch miRNA miRNA sequence (3′→5′) SEQ ID NO. 234AGUCGACGAGUAAACCAGAGU 252 MRT3847_253879C.2 153-173ucagcugcucaucuguucuca 2.5 2 253 MRT3847_54392C.5 402-422ccagcugcucauuuggucacu 2.5 3 254 MRT3847_41382C.3 118-138ucagcucuucuuuuggucucu 2.5 4 255 MRT3847_319840C.1 408-428ucagcuacugaucuggucuca 3 3 256 MRT3847_326146C.1 117-137ucagcuguuccuuuguucucu 3 4 257 MRT3847_39543C.6 768-788ucagcuguuccuuuguucucu 3 4 258 MRT3847_253942C.4 1837-1857guagcuucucacuuggucuua 3 5 259 MRT3847_260486C.4 124-144uuagcugcuucuucggucucu 3 5 260 MRT3847_210520C.2 357-377uuagaugcuuguuuggucuuu 3 6 miRNA miRNA sequence (3′→5′) SEQ ID NO. 237ACUCUUGUACCCCUCGAAGAU 261 MRT3847_303349C.1 435-455ugagaacauggggagccucua 1.5 1 262 MRT3847_14593C.6 1133-1153agaggacauggggagauucua 2 3 263 MRT3847_241913C.3 1111-1131agaggacauggggagguucua 2 3 264 MRT3847_32439C.4 1142-1162ugagaacaugggaaucuucua 2.5 2 265 MRT3847_187197C.5 689-709aaagaacauggggagccucua 2.5 3 266 MRT3847_33448C.5 1047-1067ugagaacaugggggauuucua 2.5 3 267 MRT3847_39693C.6 305-325ugugaagguggggagcuucuu 2.5 4 268 MRT3847_50432C.5  89-109ggagaacaugcagagcuucug 2.5 4 269 MRT3847_95417C.1 308-328ugagaaacuggggagcuuuuc 2.5 4 270 MRT3847_115705C.2  82-101ugagaac-uggugagcuucug 3 3 miRNA miRNA sequence (3′→5′) SEQ ID NO. 237ACUCUUGUACCCCUCGAAGAU 271 MRT3847_182667C.1 143-162ugaguac-uggggagcuucuc 3 3 272 MRT3847_184995C.1  16-36 ugagagcauggguaacuucua 3 3 273 MRT3847_253437C.4 141-160ugagcac-uggggagcuucuc 3 3 274 MRT3847_293395C.2 294-313ugagcac-uggggagcuucuc 3 3 275 MRT3847_63512C.6 321-340ugagcac-uggggagcuucuc 3 3 276 MRT3847_64829C.6 1087-1107ugagaacaugggaacuuucua 3 3 277 MRT3847_80470C.3  15-35 ugagagcauggguaacuucua 3 3 278 MRT3847_136444C.5 312-332ugagaaccugguaagcuucug 3 4 279 MRT3847_231576C.1 360-380ugagaacaucgaaagcuucuu 3 4 280 MRT3847_263317C.1  90-110ugaggacaaggggagcuuaug 3 4 281 MRT3847_304409C.1 217-237cuaaaacauggggagcuucuu 3 4 282 MRT3847_247682C.3 1287-1307ugaggaaauagggaguuucug 3 5 283 MRT3847_251048C.2 280-300ugagaacauagugaguuuuuu 3 5 284 MRT3847_270705C.2 575-595uaggaucguggggagcuucuc 3 5 285 MRT3847_304509C.2 592-612uaggaucguggggagcuucuc 3 5 286 MRT3847_62576C.4 540-560uaggaucguggggagcuucuc 3 5 287 MRT3847_67153C.3 661-681gaugaauauggggaguuucua 3 5 miRNA miRNA sequence (3′→5′) SEQ ID NO. 240CUCCGUUUCUAUGGGCGUUGU 288 MRT3847_106868C.2 318-338ggggcaaggacauccgcaacg 2.5 5 289 MRT3847_307036C.1 171-191aaggcaaaguugcccgcgacg 2.5 5 290 MRT3847_308816C.2 719-739gaggcaaagaugcgagcaacg 3 4 291 MRT3847_6248C.3 584-604gcggcaaagauacucacaacc 3 4 292 MRT3847_104943C.2 177-197aacgcaaagagaccuguaaca 3 5 293 MRT3847_290510C.2 181-201aaggcaaagaugccagcgacg 3 5 294 MRT3847_294184C.2 1090-1110gagccaaagagacccgugacg 3 5 295 MRT3847_321797C.1 847-867aaggcauagauagucgcagca 3 5 296 MRT3847_63653C.5 1096-1116aaggcaaagaugccagcaaug 3 5 297 MRT3847_9362C.2 481-501uagggaaagauacauguaaca 3 5 298 MRT3847_112761C.3 331-351gaggcaaaguuguucgcaaug 3 6 299 MRT3847_249731C.3 515-535caggcaaagaugucugcaauu 3 6 300 MRT3847_313052C.1 253-273uagguauggauacuugcaaca 3 6 301 MRT3847_318082C.1 123-143aaggcaaagcugcccgcgaug 3 6 miRNA miRNA sequence (3′→5′) SEQ ID NO. 243AGUCUCCGCUUCUGUGAGCGU 302 MRT3847_160536C.3 182-202ucaggggaggagacacucgca 2 3 303 MRT3847_290017C.2 304-324uuagaggcaaagacacucguc 2 4 304 MRT3847_97323C.1  55-75 ucagaggagaagauacucgug 2 4 305 MRT3847_182887C.1  43-63 ucagaggagaagacacgcgca 2.5 2 306 MRT3847_290275C.2 177-197ucagaggggaagacacacgcu 2.5 3 307 MRT3847_296312C.2 155-175ucagaggggaagacacacgcu 2.5 3 308 MRT3847_292252C.2 171-191ucagaggugaggacacacgcu 2.5 4 309 MRT3847_206250C.1 306-326ccagaggcggaugcauucgca 2.5 5 310 MRT3847_240825C.3 436-456acagaggcagggacacuugca 2.5 5 311 MRT3847_250458C.2 776-796gcagaggugaagaagcuugca 2.5 5 312 MRT3847_36461C.4  87-107uuagaggagaggauacucgcg 2.5 5 313 MRT3847_48749C.4 715-735gcagaggugaagaagcuugca 2.5 5 314 MRT3847_97362C.3 566-586ucagaggcaaagauacccgca 3 3 315 MRT3847_20647C.2 143-163uuagaggggaagacacgcgcu 3 4 316 MRT3847_219382C.1 147-167ucagaggggaagacacccgug 3 4 317 MRT3847_243196C.3  73-93 ucagaggcuaagagacuugua 3 4 318 MRT3847_248880C.3 760-780ucagaggggaagacacgcgug 3 4 319 MRT3847_25201C.4 173-193ucagaggggaagacacccgug 3 4 320 MRT3847_264555C.4 212-232ucagaggggaagacacacguu 3 4 321 MRT3847_28447C.6 142-162ucagaggggaagacacacguu 3 4 322 MRT3847_32431C.4  59-79 ucaggggugaagacacacgua 3 4 323 MRT3847_99342C.1 116-136ucagaggggaagacacccgug 3 4 324 MRT3847_210811C.2 273-293ucagaaacgaagacgcucguu 3 5 325 MRT3847_240622C.2  92-112uccgaggggaagauacucguu 3 5 326 MRT3847_254863C.2 175-195uccgaggggaagauacucguc 3 5 327 MRT3847_255345C.3 113-133uccgaggggaagauacucguc 3 5 328 MRT3847_257424C.1 378-398gcagaggcuguggcacucgca 3 5 329 MRT3847_38012C.4  56-76 uuagaggcgaggacacacguu 3 5 330 MRT3847_6951C.6 306-326uccgaggagaagauacucguu 3 5 331 MRT3847_263266C.4 163-183ucaguggcgaaggcguucguc 3 6 332 MRT3847_272810C.2 502-522uuagaggugauggcacucgug 3 6 miRNA miRNA sequence (3′→5′) SEQ ID NO. 246AUCCUUACCCACCUUAGCCGUU 333 MRT3847_302750C.1 259-280ggggaauggguggaaacggcaa 1.5 3 334 MRT3847_136115C.3 661-682ugggaaugggugggauggguaa 2.5 4 335 MRT3847_235247C.2 694-715ugggaaugggugggauggguaa 2.5 4 336 MRT3847_21031C.3 1364-1385auggaacugguggaauuggcaa 2.5 5 337 MRT3847_297070C.2 280-301cgggaaagguuggaauuggcaa 2.5 5 338 MRT3847_248343C.3 392-413uaggaauggguggauuuugcaa 3 3 339 MRT3847_207469C.2  1-20   ggaauggguggcgugggcaa 3 5 340 MRT3847_216295C.4 537-558caggaaaggggggaguuggcaa 3 5 341 MRT3847_287795C.2 141-162uagcaauggguuggaucgguga 3 5 342 MRT3847_302511C.2  35-56 guugaauggguggaauuggaaa 3 5 343 MRT3847_312620C.1  46-67 guugaauggguggaauuggaaa 3 5 344 MRT3847_20416C.2 679-700aaggaauugggggaauugguac 3 6 345 MRT3847_297209C.1 289-310cacgaguggggggaaucggcgg 3 6 346 MRT3847_6639C.4 195-216guggaauggguggucuugguaa 3 6

Example 22

This example describes a recombinant DNA construct of the invention,including a promoter, a terminator, transcribable sequence between thepromoter and the terminator, and at least one gene suppression elementthat is 3′ to the terminator. More specifically, this exampledemonstrates that a gene suppression element 3′ to a terminator wastranscribed and silenced a target gene in a plant cell.

Most expression cassettes include both a promoter and a terminator (i.e., a genetic element containing sequences necessary for polyadenylationof the primary transcript), between which is contained the sequence(s)to be expressed in a cell. Nonetheless, it is likely that the primarytranscript extends beyond the terminator element. In plants, it isbelieved that transcription continues some distance beyond thepolyadenylation signal and site. In one of the few studies to examinetranscription termination in plants, transcripts terminated downstreamof the polyA site by as much as 300 bp; no single transcriptionaltermination sites were found, but rather a series of potentialtermination sites that corresponded with T-rich sequences; see Hasegawaet al. (2003) Plant J., 33:1063-1072. It is believed thatpolyadenylation pathway genes are conserved from animals to plants; seeYao et al. (2002) J. Exp. Bot., 53:2277-2278. Plant mRNAs analogoussequences are found in positions similar to those of animal AAUAAA andU-rich sequences, suggesting an equivalent regulatory mechanisms inplants; see Graber et al. (1999) Proc. Natl. Acad. Sci. USA,96:14055-14060. In yeast and animals, transcripts have been shown toextend over 1 kilobase downstream of the polyadenylation signal andsite; see Proudfoot (2004) Curr. Opin. Cell Biol., 16:272-278. The 3′end of a mature RNA transcript is formed by cleavage and polyadenylationat the polyA site. Although the primary transcript extends well beyondthe polyA site, most current models for transcriptional terminationinvoke a coupling between polyadenylation and termination; see Proudfoot(2004) Curr. Opin. Cell Biol., 16:272-278. For example, some evidenceindicates that the presence of PolII “pause sites” downstream of thepolyadenylation site. Removal of such pause sites is expected to allowtranscription to extend even further downstream of the polyadenylationsite. Thus, a single RNA transcript can be used to both express a gene(with sequence upstream of the terminator) and suppress a gene (with RNAdownstream of the terminator), and furthermore allows the expression andsuppression to be temporally and spatially coupled. In one non-limitingexample, the coordinated expression of a bacterial cordapA gene andsuppression of the endogenous LKR-SDH gene has been shown to result inelevation of lysine levels in the maize kernel. Another example is theexpression in a transgenic plant of a gene encoding a Bacillusthuringiensis insecticidal protein and the production of dsRNAtargetting an essential corn rootworm (CRW) gene, the combination ofwhich provides enhanced control of CRW.

Various non-limiting embodiments are depicted in FIG. 25, where genesuppression elements can be any of those disclosed herein, e. g., thegene suppression elements depicted in FIG. 8, as well as aptamers orriboswitches. In one embodiment, an inverted repeat of at least 21 basepairs is positioned 3′ to a terminator, e. g., downstream of a typicalgene expression cassette that includes a promoter, a sequence to beexpressed, and a terminator. In other embodiments, tandem repeats ofanti-sense or sense sequence of the target gene are used as the genesuppression element. In some embodiments, the gene suppression elementis embedded in an intron directly or substantially directly 3′ to theterminator. The downstream sequence can contain a gene suppressionelement designed to be processed by a trans-acting siRNA mechanism, e.g., sequences corresponding to a target gene fused to a miRNA targetsequence, such that miRNA-triggered dsRNA production occurs resulting insilencing of a target gene. A second terminator can be included as shownin FIG. 26A, or can be omitted, as the absence of a polyadenylationsignal downstream of a gene suppression element does not reducesuppression efficiency (see Example 1) and can enhance it. Where twoterminators are included, it is preferable that the two terminators beunrelated to reduce the possibility of recombination between them.

The constructs depicted in FIG. 26A (suppression construct) and FIG. 26B(control construct) were tested in a maize protoplast assay as describedin Examples 1 and 2. Firefly luciferase suppression experiments wereperformed, and the target gene, firefly luciferase, was suppressed by aninverted repeat 3′ to the terminator, as indicated by the logarithm ofthe ratio of firefly luciferase to Renilla luciferase, “log(Fluc/Rluc)”,as depicted in FIG. 26C.

Example 23

This non-limiting example illustrates the transgenic plants of theinvention, which have in their genome recombinant DNA includingtranscribable DNA including DNA that transcribes to an RNA aptamercapable of binding to a ligand. One application of the invention is toprovide a ligand-activated, herbicide-resistant system for gene identitypreservation (“gene lock”) as well as to maintain herbicide-resistantvolunteer control.

In one embodiment, the DNA sequence encoding an “on” riboswitch isinserted into an expression cassette containing as the target sequence“CP4”, a selectable marker conferring glyphosate resistance, epsps-cp4(5-enolpyruvylshikimate-3-phosphate synthase from Agrobacteriumtumefaciens strain CP4), to conditionally express CP4 in transgenicplants. See the construct depicted in FIG. 28A, where CP4 is the targetsequence (“TS”), and FIG. 28F, which depicts a non-limiting example of aCP4 expression cassette useful for Agrobacterium-mediated transformationof maize and other crop plants, and the expected “ligand A”-controlledCP4 expression. Transgenic plants harboring the riboswitch-controlledCP4 cassette express CP4 only in the presence of the ligand, which isapplied (e. g., by a foliar spray) to the plant by means of aproprietary glyphosate formulation containing the ligand. Uponapplication, the formulated glyphosate herbicide activates CP4transcription/translation and renders the transgenic plant resistant toglyphosate. Transgenic plants are susceptible to generic glyphosateformulations that do not contain the ligand. Similarly, this approachcan be applied to any other herbicide-resistance gene/herbicidecombinations, for example, dicamba-degrading-oxygenase/dicamba, orantibiotic-resistance gene/antibiotic combination.

Ligand-activated herbicide resistance riboswitches allow formulation ofcrop-specific herbicides, by using a riboswitch that binds to adifferent ligand for selected plant species. For example, where anadenine-binding riboswitch is used for soybeans and a lysine-bindingriboswitch is used for corn, a lysine-containing glyphosate formulationwill control non-transgenic weeds as well as glyphosate-resistantsoybean volunteers (e. g., from a previous crop).

In another embodiment, an autoinduced riboswitch is used to treat seeds.If the residual herbicide lasts longer than the ligand in plant tissuesafter the ligand-containing herbicide formulation is applied, it couldcause crop damage due to the shut down of the herbicide resistance gene.One approach to prevent this is to choose a ligand that is anendogenously produced metabolite and to include a mechanism for theligand's production with the riboswitch. This makes it possible toengineer an autoinduced riboswitch to maintain expression of theherbicide resistance gene. Using a lysine-autoinduced riboswitch forglyphosate resistance as an example (FIG. 28C), the addition of a secondgene, Corynebacterium DHDPS or cordapA (“dapA”) (see U.S. Pat. Nos.6,459,019 and 5,773,691 and U.S. Patent Application Publication No.2003/0056242, which are incorporated by reference), maintains apersistent lysine level sufficient to maintain expression of both CP4and dapA. This autoinduced system also allows the ligand to be appliedby seed treatment as an alternative to including the ligand in theglyphosate formulation. Untreated seeds are viable but require treatmentwith the appropriate ligand prior to planting in order for the resultingplants to be resistant to the herbicide.

Example 24

This non-limiting example further illustrates the transgenic plants ofthe invention. One embodiment of the invention is to use an herbicidesuch as glyphosate as a chemical hybridization agent. This embodimententails transgenic plants having lower CP4 expression in male tissuesrelative to the rest of plants, whereby, when the transgenic plants areexposed to glyphosate, male sterility ensues. One approach is to combinea transcriptional control riboswitch with tissue specific control ofexpression of that riboswitch. An example is depicted in FIG. 28E, where“Promoter1” is a constitutive promoter driving expression of the targetgene (“TS”) CP4, and “Promoter 2” is a male-specific promoter drivinglysine-induced, riboswitch controlled expression of a gene suppressionconstruct for suppressing CP4 (“TS_(sup)”). Application of lysine andglyphosate (e. g. as a spray) results in male sterility. Alternatively,using the construct shown in FIG. 28D, where “Promoter 1” isconstitutive, “Promoter 2” is male-specific, and the target gene (“TS”)is CP4, initial lysine application reduces overall CP4 expression, butCP4 expression is enhanced in male tissues, thereby causing male tissuesto be more susceptible to glyphosate.

Example 25

This non-limiting example further illustrates the transgenic plants ofthe invention. One embodiment of the invention is induced expression ofa trait gene under the control of a constitutive promoter. The insertionof a riboswitch allows the trait genes, though under the control of aconstitutive promoter, to be expressed only upon selected conditions.This makes it possible to avoid yield penalty (e. g., loss of yield dueto non-selective expression of the trait gene), transgene silencing, orother concerns caused by constitutive expression of the transgenes.Non-limiting examples of such riboswitches include a glyphosate “on”riboswitch for CP4 expression, a salicylic acid “on” riboswitch fordisease resistance genes, a jasmonic acid “on” riboswitch for insectresistance genes, an ascorbate “on” riboswitch for oxidative stresstolerance genes, and a proline or glycine betaine or mannitol riboswitchfor drought tolerance genes.

Example 26

This example further illustrates the transgenic plants of the invention.One embodiment is chemically inducible or suppressible male sterility orfertility for hybridization. Preferred examples use a riboswitchcontaining an aptamer that binds a ligand that is an already registeredsubstance, e. g., an approved herbicide. In a non-limiting example, atransgenic plant harboring a male sterility gene under the control of amale-specific promoter and a glyphosate “off” riboswitch is male-sterileunless glyphosate is applied. In contrast, a transgenic plant harboringa male sterility gene under the control of a male-specific promoter anda glyphosate “on” riboswitch is male-sterile only when glyphosate isapplied.

Example 27

This non-limiting example further illustrates the transgenic plants ofthe invention. One embodiment of the invention includes artificialriboswitches that are engineered in vitro to permit expression (orsuppression) of a target sequence under inducible conditions or inresponse to biotic or abiotic stress. Such riboswitches use novelaptamers designed for a specific ligand by means well known in the art.See, for example, the detailed discussion above under the heading“Aptamers”. Especially useful riboswitches are designed to be triggeredby registered agricultural chemicals (e. g., glyphosate, dicamba),disease-induced compounds (e. g., salicylic acid), invertebratepest-induced or wounding-induced compounds (e. g., jasmonic acid), waterstress-induced compounds (e. g., proline, glycine betaine, mannitol) andoxidative stress-induced compounds (e. g., ascorbate).

Example 28

This non-limiting example further illustrates the transgenic plants ofthe invention. Riboswitches useful in transgenic plants of the inventionare designed to function at a given concentration of the ligand. Oneembodiment is a lysine riboswitch engineered to function in a transgenicplant.

Naturally occurring bacterial lysine riboswitches exist as both “on” and“off” riboswitches, and have a K_(d)˜1 millimolar (128 ppm) in vitro(see Sudarsan et al. (2003) Genes Dev., 17:2688-2697, which providesindividual and consensus sequences of prokaryotic lysine-responsiveriboswitches, and is incorporated by reference). However, maize tissuesgenerally have a lysine content of less than 50 ppm, which is thus aconcentration useful as the default state for novel lysine riboswitches.Using bacterial lysine riboswitches as an example, a series ofconstructs (FIG. 28) is transformed into maize callus, producingtransgenic maize callus lines or transgenic maize plants useful forstudying riboswitch efficacy in plants and plant cells. In someembodiments, a non-lysine-feedback-inhibited lysine biosynthetic gene,cordapA, is co-expressed in order to obtain autoinducible control ofgene expression. As shown in FIG. 28A and FIG. 28B, transcribable DNAfragments of ˜150 base pairs, encoding lysine “on” (or “off”)riboswitches, are inserted between the promoter and the target sequence(“TS”), in this example a green fluorescent protein (GFP) reporter gene.Other reporter genes or marker genes, as well as any gene of interest,can be used as the target sequence. The callus lines transformed withthese constructs display a lysine inducible (or lysine-suppressible) GFPexpression phenotype (FIG. 29, top panel, A and B). In some embodiments,a second cassette containing cordapA (“dapA”) under the control of alysine “on” riboswitch, is added (FIG. 28C and FIG. 28D); these calluslines or transgenic plants become autoinducible or autosupressible (FIG.29, top panel, C and D). FIG. 29, lower panel, schematically depicts anexpression cassette, useful in Agrobacterium-mediated transformation ofmaize and other plants, containing a lysine “on” riboswitch, wherebybinding of lysine to the aptamer of the riboswitch induces expression ofCP4 (for glyphosate resistance) as well as expression of CorynebacteriumDHDPS or cordapA (“DHDPS”). The resulting endogenous synthesis ofadditional lysine maintains expression of the transgenes.

Example 29

This example further illustrates the transgenic plants of the invention.One preferred embodiment is a transgenic plant including in its genometranscribable DNA that transcribes to a “trans”-acting riboswitch, i.e., a riboswitch that affects expression of a target sequence to whichit is not operably linked.

In some embodiments, the “trans” riboswitch is flanked by ribozymes (e.g., self-splicing or hammerhead ribozymes) and is transcribed under thecontrol of a pol II promoter (FIG. 27A); see, for example, Bayer andSmolke (2005) Nature Biotechnol., 23:337-343, which is incorporated byreference. In other embodiments, the “trans” riboswitch is transcribedunder the control of a pol III promoter (FIG. 27B), wherebytranscription is terminated at a poly-T region. In other embodiments,the “trans” riboswitch is flanked by intron-splicing junctions (FIG.27C), whereby the riboswitch is spliced out after transcription; suchembodiments can optionally include DNA that transcribes to a microRNArecognition site or DNA that transcribes to RNA capable of formingdouble-stranded RNA (dsRNA) (FIG. 27D). Embodiments containingintron-embedded transcribable DNA can optionally include one or moregene expression (or suppression) elements (“GOI” in FIG. 27C and FIG.27D). Alternatively, the transcribed riboswitch can be flanked bydouble-stranded RNA that can be cleaved through an RNAi (siRNA or miRNA)processing mechanism (FIG. 27E). In yet other embodiments, the “trans”riboswitch is flanked by DNA that transcribes to a microRNA recognitionsite (FIG. 27E), whereby cleavage of the transcribed riboswitch occursafter binding of the corresponding mature miRNA to the miRNA recognitionsite. These approaches enable the creation of noncoding riboregulatorswith defined 5′ and 3′ ends that are free of potentially interferingflanking sequences. In still other embodiments, the “trans” riboswitchis flanked by DNA that transcribes to RNA capable of formingdouble-stranded RNA (dsRNA) (FIG. 27E). In some of these cases, thedsRNA is processed by an RNAi (siRNA or miRNA) mechanism, whereby thetranscribed riboswitch is cleaved from the rest of the transcript. Inother cases, the two transcribed RNA regions flanking the “trans”riboswitch form at least partially double-stranded RNA “stem” betweenthemselves, wherein the “trans” riboswitch serves as a “spacer” or“loop” in a stem-loop structure.

In one example, the transgenic plant has in its genome an expressioncassette using pol II promoters to express a “trans” riboswitch flankedby self-cleaving hammerhead ribozyme sequences, resulting in ariboswitch with defined 5′ and 3′ ends, free of potentially interferingflanking sequences (FIG. 27A; also see Bayer and Smolke (2005) NatureBiotechnol., 23:337-343). An alternative approach uses expressioncassettes under the control of pol III promoters to produce non-codingRNAs with minimal 5′ and 3′ flanking sequences. RNA polymerase IItranscribes structural or catalytic RNAs that are usually shorter than400 nucleotides in length, and recognizes a simple run of T residues asa termination signal; it has been used to transcribe siRNA duplexes (Luet al. (2004) Nucleic Acids Res., 32:e171, which is incorporated byreference). Riboregulators expressed by Pol III are expected to generatetranscripts with relatively defined 5′ and 3′ ends (FIG. 27B). It hasbeen used to transcribe siRNA duplexes. Alternatively, a “trans”riboswitch is fused to the minimal sequences required for splicing, andendogenous intron splicing mechanisms are used to release theriboregulator (FIG. 27C). This intron-embedded configuration providesthe advantage of allowing concurrent expression of a gene of interest(GOI).

One specific application of “trans” riboswitch is their use ingenerating transgenic plants with inducible male sterility or fertility.Hybrid plant varieties have a significant yield advantage over theirinbred counterparts, but can be more costly to produce. Reversible malesterility/fertility is one of the most cost-effective ways to producehybrids. In this application, “trans” riboswitches are designed totarget endogenous genes required for male development. Suppression ofany of these genes results in male sterility. “Trans” riboswitchesdriven by male-specific pol II promoters (FIG. 27A) can be used tocontrol the expression of any target sequence or gene that leads to celldeath (apoptosis) or growth arrest. Alternatively “trans” riboswitchestranscribed under the control of pol III promoters (FIG. 27B), which areconstitutive, are designed to be male specific to avoid undesirablephenotypes. In an inducible male fertility system, the “trans”riboswitch used is an “off” switch (where the riboswitch is bound to itstarget sequence by default and is released from the target sequence whenbound by ligand), supply of the ligand (by endogenous biosynthesis orexogenous application, e. g., by spraying) restores fertility. In aninducible male sterility system, the “trans” riboswitch used is an “on”switch, and binding of the ligand results in the “trans” riboswitchbinding to its target sequence and inducing male sterility.

Another specific application of “trans” riboswitches is their use ingenerating transgenic plants displaying “gene lock”. Seeds containing an“off” “trans” riboswitch designed to target endogenous genes requiredfor germination will not be able to germinate. When under the control ofa pol II promoter (FIG. 1A), any gene functioning in cell death orgrowth arrest can be targetted. Alternatively Pol III driven “trans”riboswitches (FIG. 1B) would have to target genes that are specific togermination to avoid undesirable phenotypes. The germination restorationcould be seed treatment and illegally copied seeds without seedtreatment would not be able to germinated.

“Trans” riboswitches, similarly to the “cis” riboswitches described inExamples 23 through 2, are useful in regulating transgenes. In aspecific example, a transgenic plant including a “trans” riboswitchdesigned to regulate the glyphosate-resistance transgene CP4 as thetarget sequence is particularly useful in “trans” riboswitch-controlledapplications parallel to that described in Example 23 (glyphosate as aligand for ligand-activated herbicide resistance, or for control ofherbicide resistant volunteers) and Example 24 (glyphosate as a chemicalhybridization agent). To illustrate this approach, CP4 expression issuppressed in stably transformed maize callus. A modified transcribableDNA encoding an “off” “trans” riboswitch with theophylline as its ligand(Bayer and Smolke (2005) Nature Biotechnol., 23:337-343) is designed totarget CP4 as a target sequence. Transcription of the theophyllineriboswitch can be driven either by a pol II promoter (e. g., FIG. 27A)or by pol III promoter (e. g., FIG. 27B). The transcribable DNA isinserted into a binary vector (FIG. 30) and co-transformed into maizecallus under nptIII selection, generating stably transformed maizecallus lines. CP4 expression is assayed in the transformed cells, whereCP4 expression is observed to be suppressed in transformed cells thatare treated with theophylline.

Similarly, a “trans” riboswitch is used to control expression of anendogenous target sequence (lysine ketoglutarate reductase/saccharopinedehydrogenase gene, LKR/SDH) in stably transformed maize plants. Amodified transcribable DNA encoding an “off” “trans” riboswitch withtheophylline as its ligand (Bayer and Smolke (2005) Nature Biotechnol.,23:337-343) is designed to target at least one region of the LKR/SDHsequence, and co-transformed into maize callus. LKR/SDH expression isassayed in the resulting transformed cells, where LKR/SDH expression isobserved to be suppressed in transformed cells that are treated withtheophylline.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations can be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1-61. (canceled)
 62. A recombinant DNA construct comprising atranscribable engineered miRNA precursor designed to suppress a targetsequence, wherein said transcribable engineered miRNA precursor isderived from the fold-back structure of a maize or soybean MIR sequenceselected from the group consisting of the MIR sequences identified inTables 3, 4, 5, 6, 9, and 10 and their complements.
 63. A method tosuppress expression of a target sequence in a plant cell, comprisingtranscribing in a plant cell the recombinant DNA construct of claim 62,whereby expression of said target sequence is suppressed relative to itsexpression in the absence of transcription of said recombinant DNAconstruct. 64-85. (canceled)
 86. A transgenic seed having in its genomethe recombinant DNA construct of claim
 62. 87. A transgenic plant grownfrom the transgenic seed of claim
 86. 88. The transgenic seed of claim86, wherein said transgenic seed has modified primary metabolite, traceelement, carotenoid, vitamin, or secondary metabolite composition, orimproved storage or processing quality.
 89. The transgenic plant ofclaim 87, wherein said transgenic plant has at least one altered trait,relative to a plant lacking said recombinant DNA construct, selectedfrom the group of traits consisting of: (a) improved abiotic stresstolerance; (b) improved biotic stress tolerance; (c) improved resistanceto a pest or pathogen of said plant; (d) modified primary metabolitecomposition; (e) modified secondary metabolite composition; (f) modifiedtrace element, carotenoid, or vitamin composition; (g) improved yield;(h) improved ability to use nitrogen or other nutrients; (i) modifiedagronomic characteristics; (j) modified growth or reproductivecharacteristics; and (k) improved harvest, storage, or processingquality.
 90. A transgenic plant cell having in its genome a recombinantDNA construct comprising a transcribable engineered miRNA precursordesigned to suppress a target sequence, wherein said transcribableengineered miRNA precursor is derived from the fold-back structure of amaize or soybean MIR sequence selected from the group consisting of theMIR sequences identified in Tables 3, 4, 5, 6, 9, and 10 and theircomplements.
 91. The transgenic plant cell of claim 90, wherein saidrecombinant DNA construct further comprises a gene expression elementfor expressing at least one gene of interest.
 92. A regeneratedtransgenic plant or seed prepared from the transgenic plant cell ofclaim
 90. 93. A method of providing at least one altered plant tissue,comprising: (a) providing a transgenic plant comprising a regeneratedplant prepared from a transgenic plant cell of claim 90, or a progenyplant of said regenerated plant; and (b) transcribing said recombinantDNA construct in at least one tissue of said transgenic plant, wherebyan altered trait in said at least one tissue results, relative to tissuewherein said recombinant DNA construct is not transcribed, said alteredtrait being selected from: (i) improved abiotic stress tolerance; (ii)improved biotic stress tolerance; (iii) improved resistance to a pest orpathogen of said plant; (iv) modified primary metabolite composition;(v) modified secondary metabolite composition; (vi) modified traceelement, carotenoid, or vitamin composition; (vii) improved yield;(viii) improved ability to use nitrogen or other nutrients; (ix)modified agronomic characteristics; (x) modified growth or reproductivecharacteristics; and (xi) improved harvest, storage, or processingquality.
 94. The method of claim 93, wherein said transgenic plant is atransgenic crop plant.